Modular aptamer-regulated ribozymes

ABSTRACT

An extensible RNA-based framework for engineering ligand-controlled gene regulatory systems, called ribozyme switches, that exhibit tunable regulation, design modularity, and target specificity is provided. These switch platforms typically contain a sensor domain, comprised of an aptamer sequence, and an actuator domain, comprised of a hammerhead ribozyme sequence. A variety of modes of standardized information transmission between these domains can be employed, and this application demonstrates a mechanism that allows for the reliable and modular assembly of functioning synthetic hammerhead ribozyme switches and regulation of ribozyme activity in response to various effectors. In some embodiments aptamer-regulated cis-acting hammerhead ribozymes are provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. Ser. No.11/938,220, filed Nov. 9, 2007, now U.S. Pat. No. 8,158,595, whichclaims the benefit of and priority to U.S. Provisional Application No.60/857,824 filed on Nov. 9, 2006, and U.S. Provisional Application No.60/875,774 filed on Dec. 19, 2006, the contents of which applicationsare incorporated herein by reference in their entirety.

STATEMENT REGARDING FEDERALLY SPONSORED R & D

This invention was made with government support under Grant No.W911NF-05-1-0281 awarded by the United States Army Research Office andunder Grant No. GM074767 awarded by the National Institute of Health.The government has certain rights in the invention.

BACKGROUND OF THE INVENTION

Basic and applied biological research and biotechnology are limited byour ability to get information into and out from living systems, and toact on information inside living systems. Endy (2005) Nature 438:449-53;Voigt (2006) Curr Opin Biotechnol 17:548-57; and Kobayashi et al. (2004)Proc Natl Acad Sci USA 101:8414-9. For example, there are only a smallnumber of inducible promoter systems available to provide control overgene expression in response to exogenous molecules. Gossen et al. (1992)Proc Natl Acad Sci USA 89:5547-51; and Lutz et al. (1997) Nucleic AcidsRes 25:1203-10. Many of the molecular inputs to these systems are notideal for broad implementation, as they can be expensive and introduceundesired pleiotropic effects. In addition, broadly-applicable methodsfor getting information out of cells non-invasively have been limited tostrategies that rely on protein and promoter fusions to fluorescentproteins, which enable researchers to monitor protein levels andlocalization and transcriptional outputs of networks, leaving asignificant amount of the cellular information content currentlyinaccessible.

To address these challenges scalable platforms are needed for reportingon, responding to, and controlling any intracellular component in aliving system. A striking example of a biological communication andcontrol system is the class of RNA regulatory elements calledriboswitches, comprised of distinct sensor and actuation (generegulatory) functions, that control gene expression in response tospecific ligand concentrations. Mandal et al. (2004) Nat Rev Mol CellBiol 5:451-63. Building on these natural examples, engineered riboswitchelements have been developed for use as synthetic ligand-controlled generegulatory systems. Kim et al. (2005) RNA 11:1667-77; An C I et al.(2006) RNA 12:710-6; Bayer et al. (2005) Nat Biotechnol 23:337-43; andIsaacs et al. (2006) Nat Biotechnol 24:545-54. However, as versatile asthese early examples of riboswitch engineering are, there are additionalchallenges posed, such as greater ease in portability across organismsand systems, and improved modularity and component reuse.

There is a need, therefore, to develop a universal and extensibleRNA-based platform that will provide a framework for the reliable designand construction of gene regulatory systems that can control theexpression of specific target genes in response to various effectormolecules.

SUMMARY OF THE INVENTION

Implementing five engineering design principles (DPs) in addressing thechallenge of developing a universal and extensible RNA-based platform,this application describes an extensible RNA-based framework forengineering ligand-controlled gene regulatory systems, called ribozymeswitches, that exhibit tunable regulation, design modularity, and targetspecificity. In particular, the subject ribozyme switches address:scalability (DP1: a sensing platform enabling de novo generation ofligand-binding elements for implementation within the sensor domain);portability (DP2: a regulatory element that is independent ofcell-specific machinery or regulatory mechanisms for implementationwithin the actuator domain); utility (DP3: a mechanism through which tomodularly couple the control system to functional level components);composability (DP4: a mechanism by which to modularly couple theactuator and sensor domains without disrupting the activities of theseindividual elements); and reliability (DP5: a mechanism through which tostandardize the transmission of information from the sensor domain tothe actuator domain).

One aspect of the invention relates to an aptamer-regulated cis-actinghammerhead ribozyme. In general this regulated ribozyme includes acis-acting hammerhead ribozyme (or “chRz”), e.g., a ribozyme comprisingof a catalytic core with stem I, stem II and stem III duplex regionsextending therefrom. Each of the stem I and stem II duplexes include asingle-stranded loop region opposite to the catalytic core, these loopsbeing referred to as loop I and loop II respectively. An example of thisstructure is shown in FIG. 1. The regulated ribozyme also includes atleast one aptamer directly coupled through an information transmissiondomain to loop I and/or loop II. The aptamer can be chosen based on itsability to bind a ligand or otherwise “sense” a change in environment(such as pH, temperature, osmolarity, salt concentration, etc) in amanner that alters the base-pairing with the information transmissiondomain that is carried over as a structural change in the hammerheadribozyme. In certain embodiments, the aptamer and informationtransmission domains are integrated such that binding of the ligand tothe aptamer causes a change in the interaction of the informationtransmission domain with one or more of the loop, the stem or thecatalytic core such that the ribozyme undergoes self-cleavage of abackbone phosphodiester bond at a rate dependent upon the presence orabsence of the ligand. In certain embodiments, the presence of ligandwill increase the rate of self-cleavage relative to the absence ofligand, while in other embodiments the rate of self-cleavage is greaterin the absence of ligand relative to the presence of ligand. In certainpreferred embodiments, the difference in the rate of cleavage is atleast 100 fold, and even more preferably 1000 or 10,000 fold.

In certain embodiments, the hammerhead ribozyme core comprises thepolynucleotide sequence 5′- . . . UCH . . . UWYGANGA . . . GAAA . . .-3′, wherein H is selected from A, C, and U; W is selected from C, U,and A; Y is selected from C and U; and N is selected from A, C, G, andU.

In certain embodiments, binding of the ligand to the aptamer alters thesize of the loop I or loop II to which the information transmissiondomain and aptamer are coupled, and thereby alters the ability of theribozyme to undergo self-cleavage in a manner dependent on the ligand,such as by altering tertiary structural contacts involving the loop towhich the information transmission domain and aptamer are coupled.

In certain embodiments, binding of the ligand to the aptamer alters thesize of the catalytic core, thereby altering the ability of the ribozymeto undergo self-cleavage in a manner dependent on the ligand.

In certain embodiments, binding of the ligand to the aptamer can causehelix-slipping involving the information transmission domain.

In certain embodiments, binding of the ligand to the aptamer causesstrand-displacement involving the information transmission domain.

In certain embodiments, a single information transmission domain andaptamer are coupled to loop I, while in others a single informationtransmission domain and aptamer are coupled to loop II. However, thepresent invention contemplates ribozymes having 2 or more (e.g., 2, 3,4, 5) information transmission domain/aptamers coupled through loops Iand II. For instance, the ribozyme can be engineered to have a firstaptamer coupled to loop I through a first information transmissiondomain and a second aptamer coupled to loop II through a secondinformation transmission domain. The aptamers can be chosen to bind thesame ligand, including binding to the same ligand but with differentaffinities, or the aptamers can be chosen to bind different ligands fromone another.

In certain embodiments, the aptamer is chosen to bind a small molecule,such as one having a molecular weight less than 2500 amu and/or onewhich is cell permeable. In other embodiments, the aptamer is chosen tobind a metal ion.

In certain embodiments, the aptamer is chosen to bind a ligand which isa natural product, such as a signal transduction second messengermolecule.

The aptamer can be chose to selectively bind a protein, and in certainembodiments, that selectivity can be for a post-translationally modifiedform of the protein, or the unmodified protein. The aptamer may also beselected to be able to selectively bind a particular splice variant of aprotein.

To further illustrate, the aptamer can be one which binds a ligandselected from the group consisting of polypeptides, peptides, nucleicacids, carbohydrates, fatty acids and lipids, a non-peptide hormone(such as steroids) and metabolic precursors or products thereof.

It may be an aptamer that senses a change in substrate or product of ametabolic process, such as binding a ligand selected from the groupconsisting of enzyme co-factors, enzyme substrates and products ofenzyme-mediated reactions.

In certain embodiments, the ribozymes described herein (cis or trans)can be generated using RNA or an analog thereof. In certain embodiments,the ribozyme is comprised of RNA or an analog thereof, and DNA or ananalog thereof. For instance, the ribozyme core can be RNA or an analogthereof, and the one or more of the stems of the ribozyme can be DNA oran analog thereof. Merely to illustrate, a aptamer-regulated ribozyme ofthe present invention may include at least one modified base moietywhich is selected from the group including but not limited to5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil,hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxytriethyl)uracil, 5-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil; beta-D-mannosylqueosine,5-methoxycarboxymethyluracil, 5-methoxyuracil,2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v),wybutoxosine, pseudouracil, queosine, 2-thiocytosine,5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,uracil-5-oxyacetic acid methyl ester, uracil-5-oxyacetic acid (v),5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w,and 2,6-diaminopurine.

In addition to the nucleic acid that is itself functional as theregulated ribozyme, the present invention also provides expressionconstructs that include a “coding sequence” which, when transcribed toRNA, produces the aptamer regulated cis-acting hammerhead ribozyme, andmay include one or more transcriptional regulatory sequences thatregulate transcription of that sequence in a cell containing theexpression construct.

In certain embodiments, the expression construct can be designed toinclude one or more ribozymes in an RNA transcript, such as in the 3′untranslated region (3′-UTR), so as to regulate transcription, stabilityand/or translation of that RNA transcript in a manner dependent on theligand. To further illustrate, the expression construct can include acoding sequence for a polypeptide such that the mRNA transcript includesboth the polypeptide coding sequence as well as one or more of theregulated ribozymes. In this way, expression of the polypeptide can berendered dependent on the ligand to which the aptamer binds.

The present invention also provides cells that have been engineered toinclude such expression constructs. Still another aspect of theinvention relates to methods for regulating expression of a recombinantgene. Those methods include providing such a cell, and contacting thecell with the ligand in an amount that alters the activity of theribozyme, and therefore, the expression of the recombinant gene.

Still another aspect of the invention relates to an aptamer-regulatedtrans-acting hammerhead ribozyme. In general, the regulatedtrans-ribozyme includes a trans-acting hammerhead ribozyme (or “thRz”),e.g., a ribozyme having a catalytic core, a 5′ targeting arm whichhybridizes to a 3′ sequence of a target nucleic acid, a 3′ targeting armwhich hybridizes to a 5′ sequence of the target nucleic acid, and a stemduplex region extending from the catalytic core with a single-strandedloop region opposite the catalytic core. An example of this structure isshown in FIG. 20. The ribozyme includes an aptamer and an informationtransmission domain having a first and second end, wherein theinformation transmission domain is directly coupled to the loop throughthe first end and the aptamer through said second end. The aptamer canbe chosen based on its ability to bind a ligand or otherwise “sense” achange in environment (such as temperature, pH, etc) in a manner thatalters the interaction of the information transmission domain with oneor more of the loop, the stem or the catalytic core, such that theribozyme cleaves the target nucleic acid at a rate dependent upon thepresence or absence of said ligand. In certain embodiments, the presenceof ligand will increase the rate of cleavage relative to absence of theligand, while in other embodiments the rate of cleavage is greater inthe absence of ligand relative to the presence of ligand. In certainpreferred embodiments, the difference in the rate of cleave is at least100 fold, and even more preferably 1000 or 10,000 fold.

In certain embodiments, binding of the ligand to the aptamer alters thesize of the loop, and thereby alters the ability of the ribozyme tocleave the target nucleic acid in a manner dependent on the ligand.

In certain embodiments, binding of the ligand to the aptamer alters thesize of the catalytic core, thereby altering the ability of the ribozymeto cleave the target nucleic acid in a manner dependent on the ligand.

In certain embodiments, binding of the ligand to the aptamer can causehelix-slipping involving the information transmission domain.

In certain embodiments, binding of the ligand to the aptamer causesstrand-displacement involving the information transmission domain.

In certain embodiments, the aptamer is chosen to bind a small molecule,such as one having a molecular weight less than 2500 amu and/or onewhich is cell permeable. In other embodiments, the aptamer is chosen tobind a metal ion.

In certain embodiments, the aptamer is chosen to bind a ligand which isa natural product, such as a signal transduction second messengermolecule. The aptamer can be chosen to selectively bind a protein, andin certain embodiments, that selectivity can be for apost-translationally modified form of the protein, or the unmodifiedprotein. The aptamer may also be selected to be able to selectively binda particular splice variant of a protein.

To further illustrate, the aptamer can be one which binds a ligandselected from the group consisting of polypeptides, peptides, nucleicacids, carbohydrates, fatty acids and lipids, a non-peptide hormone(such as steroids) and metabolic precursors or products thereof.

It may be an aptamer that senses a change in substrate or product of ametabolic process, such as binding a ligand selected from the groupconsisting of enzyme co-factors, enzyme substrates and products ofenzyme-mediated reactions.

In certain embodiments, the ribozymes described herein (cis or trans)can be generated using RNA or an analog thereof. In certain embodiments,the ribozyme is comprised of RNA or an analog thereof, and DNA or ananalog thereof. For instance, the ribozyme core can be RNA or an analogthereof, and the one or more of the stems of the ribozyme can be DNA oran analog thereof.

In addition to the nucleic acid that is itself functional as theregulated ribozyme, the present invention also provides expressionconstructs that include a “coding sequence” which, when transcribed toRNA, produces the aptamer regulated trans-acting hammerhead ribozyme,and may include one or more transcriptional regulatory sequences thatregulate transcription of that sequence in a cell containing theexpression construct. The present invention also provides cells thathave been engineered to include such expression constructs. Stillanother aspect of the invention relates to methods for regulatingexpression of a target gene (or other RNA species). Those methodsinclude providing such a cell wherein the trans-acting ribozyme, in itsactive form, cleave an mRNA that is transcribed in the cell, andcontacting the cell with the ligand in an amount that alters theactivity of the ribozyme, and therefore, the expression of the targetgene.

Another aspect of the invention provides a cell having a metabolicpathway of one or more reactions, and in which one or more of thesubject aptamer-regulated trans-acting hammerhead ribozymes act ascontrol elements on the metabolic pathway by inhibiting expression ofone or more target genes. In such embodiments, ligand binding to theaptamer causes a change in the trans-acting ribozyme between twoconformational states, in one of which the trans-acting ribozymeinhibits expression of a target gene and in the other of which thetrans-acting ribozyme does not inhibit expression of the target gene. Inthis embodiment, the metabolic pathway is regulated at least in part bythe activity level of the trans-acting nucleic acid, and therefore, thelevel of aptamer present. Such embodiments may be used to regulate ametabolic pathway that includes at least one reaction mediated by anenzyme, such as where the trans-acting ribozyme regulates expression ofthe enzyme.

Likewise, another aspect of the invention provides a cell having ametabolic pathway of one or more reactions, and in which one or more ofthe subject aptamer-regulated cis-acting hammerhead ribozymes act ascontrol elements on the metabolic pathway by inhibiting expression ofone or more target genes into which the cis-acting ribozymes has beenengineered so as to be part of the mRNA transcript of the gene,preferably as part of the 3′-UTR. In such embodiments, the ligandbinding to the aptamer causes a change in the cis-acting ribozymebetween two conformational states, in one of which the cis-actingribozyme inhibits expression of the target gene by undergoingself-cleavage, and in the other of which the trans-acting ribozyme doesnot cleave itself. In this embodiment, the metabolic pathway isregulated at least in part by the activity level of the cis-actingnucleic acid, and therefore, the level of aptamer present. Suchembodiments may be used to regulate a metabolic pathway that includes atleast one reaction mediated by an enzyme, such as where the cis-actingribozyme regulates expression of the enzyme.

Another aspect of the invention provides a method for renderingexpression of a target gene in a cell dependent on the presence orabsence of a ligand, by utilizing a version of the subjectaptamer-regulated trans-acting ribozyme that, in it's active form,cleaves a transcript produced by transcription of the target gene, andthereby inhibits expression of the target gene in a manner dependent onthe presence or absence of the ligand. Such embodiments can be designedto rely on ligands that are produced by the cell, or designed to rely onligands that are cell permeable agents contacted with the cell.

Likewise, the aptamer-regulated cis-acting ribozymes of the inventioncan be used to render expression of a target gene in a cell dependent onthe presence or absence of a ligand. In these embodiments, the cell isengineered with an expression construct that includes a coding sequencefor the target gene, which when transcribed to an mRNA transcript, alsoincludes one or more aptamer-regulated cis-acting ribozymes in the mRNA.Ligand binding to the aptamer causes a change in the cis-acting ribozymebetween two conformational states, in one of which the cis-actingribozyme inhibits expression of the target gene present with theribozyme in the same transcript, while in the other the cis-actingribozyme does not inhibit expression of the target gene. In this way,the cis-acting ribozyme(s) present in the transcript can regulatetranscription, stability and/or translation of the mRNA in a mannerdependent on the ligand. Such embodiments can be designed to rely onligands that are produced by the cell, or designed to rely on ligandsthat are cell permeable agents contacted with the cell.

In still another embodiment, the present invention provides a method fordetermining the amount of an analyte in a cell which expresses areporter gene by way of the cell also containing an aptamer-regulatedtrans-acting ribozyme that cleaves the reporter gene in a mannerdependent on the level of analyte. Binding of the analyte to the aptamerinduces a conformational change between active and inactive forms thetrans-acting ribozyme, and the active form inhibits expression of thereporter. The method can include measuring the amount of expression ofthe reporter gene, and correlating the amount of expression of thereporter gene with the amount of analyte, thereby determining the amountof the ligand in the cell. Exemplary reporter molecules include, withoutlimitation, fluorescent or luminescent reporter proteins such as greenfluorescent protein (GFP) or luciferase, enzymatic reporters such asalkaline phosphatase, or colorimetric reporters such as lacZ.

Likewise, in other embodiments, the present invention provides a methodfor determining the amount of an analyte in a cell which expresses areporter gene that produces an mRNA that includes one or moreaptamer-regulated cis-acting ribozyme that regulate the transcription,stability and/or translation of the mRNA transcript in a mannerdependent on the level of analyte. Binding of the analyte to the aptamercauses a change in the cis-acting ribozyme between two conformationalstates, in one of which the cis-acting ribozyme inhibits (or otherwisereduces) expression of the reporter gene and in the other of which thecis-acting ribozyme does not inhibit expression of the reporter gene.The method can include measuring the amount of expression of thereporter gene, and correlating the amount of expression of the reportergene with the amount of analyte, thereby determining the amount of theligand in the cell. Exemplary reporter molecules include, withoutlimitation, fluorescent or luminescent reporter proteins such as greenfluorescent protein (GFP) or luciferase, enzymatic reporters such asalkaline phosphatase, or colorimetric reporters such as lacZ.

Yet another aspect of the invention provides methods and compositionsfor treating or preventing infection by a pathogenic agent. Such methodsinclude administering, to a patient, a sufficient amount of anaptamer-regulated trans-acting ribozyme that cleaves a pathogen orcellular gene, e.g. required for pathogenesis, in a manner dependent onaptamer binding of a ligand that is produced as a consequence ofpathogenic infection.

Another aspect of the invention provides a method for causing phenotypicregulation of cell growth, differentiation or viability in cells of apatient. Such methods include introducing one of the subjectaptamer-regulated trans-acting ribozymes into cells of the patient. Inthese embodiments, the aptamer binds to a ligand present in the patientwhich has a concentration that is dependent on cellular phenotype.Binding of the ligand to the aptamer induces a conformational changebetween active and inactive forms of the trans-acting ribozyme, wherethe active form of the trans-acting ribozyme inhibits expression of atarget gene that alters the regulation of cell growth, differentiationor viability in the cells. The target of the ribozyme can be selected toeither induce or prevent cell death, induce or inhibit differentiation,or induce or inhibit proliferation of the cells in a manner dependent onthe presence of the ligand. In certain embodiments, the ribozyme, or anexpression construct for transcribing the ribozyme in cells, isintroduced ex vivo into cells which are then transplanted into thepatient.

Likewise, the present invention provides a method for causing phenotypicregulation of cell growth, differentiation or viability in cellstransplanted into a patient, by introducing into cells ex vivo anexpression construct that produces a transcript that includes a codingsequence for a polypeptide that causes phenotypic regulation, and one ormore of the subject aptamer-regulated cis-acting ribozymes that regulatethe level of expression of the polypeptide in a manner dependent on theconcentration of ligand, and the concentration of the ligand is in turndependent on the cellular phenotype. In such embodiments, ligand bindingto the aptamer causes a change in the cis-acting ribozyme between twoconformational states, in one of which the cis-acting ribozyme inhibitsexpression of the polypeptide and in the other of which the cis-actingribozyme does not inhibit expression of the polypeptide. Thus,expression of the polypeptide alters regulation of cell growth,differentiation or viability in the cells in a manner dependent on theconcentration of ligand.

Merely for illustration, the method can be used to prevent the growth ofhyperplastic or tumor cells, or even the unwanted proliferation ofnormal cells. It can be used to induce the death of fat cells. It canalso be used to regulate growth and differentiation of stem cells, or toregulate activation of an immune response.

Another aspect of the invention provides pharmaceutical preparations andcompositions comprising an aptamer-regulated ribozyme of the presentinvention, an expression construct which, when transcribed, produces anRNA including the ribozyme, and a pharmaceutically acceptable carriersuitable for administration use to a human or non-human patient.Optionally, the pharmaceutically acceptable carrier is selected frompharmaceutically acceptable salts, ester, and salts of such esters. Incertain preferred embodiments, the present invention provides apharmaceutical package or kit comprising the pharmaceutical preparationwhich includes at least one aptamer-regulated ribozyme and apharmaceutically acceptable carrier, in association with instructions(written and/or pictorial) for administering the preparation to a humanpatient.

Still another aspect of the present invention provides a method ofconducting a pharmaceutical business comprising: (a) identifying anaptamer-regulated trans-ribozyme which, when switched “on,” inhibitsproliferation of target cells in vivo and reduces the effects of adisorder involving unwanted proliferation of the target cells; (b)conducting therapeutic profiling of the an aptamer-regulatedtrans-ribozyme identified in step (a) for efficacy and toxicity inanimals; and (c) formulating a pharmaceutical preparation including oneor more of the aptamer-regulated trans-ribozymes identified in step (b)as having an acceptable therapeutic profile.

The method of conducting a pharmaceutical business may further comprisean additional step of establishing a distribution system fordistributing the pharmaceutical preparation for sale, and optionally,establishing a sales group for marketing the pharmaceutical preparation.

Yet still another aspect of the present invention provides a method ofconducting a pharmaceutical business comprising: (a) identifying anaptamer-regulated trans-ribozyme which, when switched “on,” inhibitsproliferation of target cells in vivo and reduces the effects of adisorder involving unwanted proliferation of the target cells; (b)(optionally) conducting therapeutic profiling of an aptamer-regulatedtrans-ribozyme identified in step (a) for efficacy and toxicity inanimals; and (c) licensing, to a third party, the rights for furtherdevelopment of the aptamer-regulated trans-ribozyme.

The skilled artisan recognizes that an aptamer-regulated trans-ribozymethat is useful for treating any disorder, including, but not limited toinhibiting pathogenic replication and/or infection, regulation of theimmune response, or modulation of the cellular state of a cell, may beused in the methods of conducting a pharmaceutical business as describedherein.

Still other aspects of the invention provide a library ofaptamer-regulated ribozymes, such as libraries having a variegatedpopulation of ribozymes having different aptamers and/or differentribozyme regions (such as targeting sequences, stem or loop sequences,as described above). These libraries may have diversity among theaptamers with respect to the types of ligands that can be bound(specificity) and/or the variation in affinity for the same ligand.

Certain embodiments are also directed to a method of establishing aconditional genetic network. The method may comprise engineering a cellto include an aptamer-regulated trans-acting ribozyme, wherein theaptamer domain is responsive to a ligand and the ribozyme domain istargeted to a gene, the expression of which is otherwise unassociatedwith a signaling, metabolic, enzymatic, or any biochemical pathway thatproduces the ligand or modulates the level of the ligand. Thus, whenswitched “on”, the ribozyme modulates expression of the gene, therebyestablishing a conditional genetic network. A conditional geneticnetwork may be useful, for example, in engineering an intracellularsignaling network.

Likewise, the aptamer-regulated cis-acting ribozymes of the inventioncan be used to establish a conditional genetic network. In theseembodiments, the cell is engineered with an expression construct thatincludes a coding sequence for the target gene, which when transcribedto an mRNA transcript, also includes one or more aptamer-regulatedcis-acting ribozymes in the mRNA. The expression of the target gene isotherwise unassociated with a signaling, metabolic, enzymatic, or anybiochemical pathway that produces the ligand or modulates the level ofthe ligand. This, when switched “on”, the ribozyme modulates expressionof the gene, thereby establishing a conditional genetic network.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. General design strategy for engineering ribozyme switches. Thearrow indicates the cleavage site. (A) General compositional frameworkand design strategy for engineering cis-acting hammerhead ribozyme-basedregulatory systems; restriction enzyme sites are underlined. (B) Modularcoupling strategies of the sensor and regulatory domains to maintain invivo activity of the individual domains.

FIG. 2. Regulatory properties of the strand displacement informationtransmission mechanism. (A) Gene expression ‘ON’ ribozyme switchplatform, L2bulge1. (B) Gene expression ‘OFF’ ribozyme switch platform,L2bulgeOff1. The theophylline-dependent gene regulatory behavior of (C)L2bulge1 (‘ON’ switch), (D) L2bulgeOff1 (‘OFF’ switch), and L2Theo(non-switch control). Gene expression levels are reported in fold asdefined in Example 2 and normalized to the expression levels in theabsence of effector.

FIG. 3. Regulatory properties of the helix slipping informationtransmission mechanism. (A) Gene expression ‘OFF’ ribozyme switchplatform based on helix slipping, L2 cm4. The base stem of the aptameris replaced with a communication module. (B) Regulatory activities ofhelix slipping-based ribozyme switches. Gene regulatory effects of the‘OFF’ switches at 5 mM theophylline are reported in fold repressionrelative to expression levels in the absence of effector. Thecorresponding communication module sequences are indicated. Geneexpression levels are reported as described in FIG. 2.

FIG. 4. Tunability of the strand displacement-based ribozyme switches.(A) Sequences targeted by the rational tuning strategies are indicatedin the dashed boxes on the effector-bound conformations of L2bulge1(ribozyme inactive) and L2bulgeOff1 (ribozyme active). Regulatoryactivities of tuned strand displacement-based (B) ‘ON’ and (C) ‘OFF’ribozyme switches. Gene regulatory effects of these switches at 5 mMtheophylline are reported in fold induction for ‘ON’ switches and foldrepression for ‘OFF’ switches relative to the expression levels in theabsence of theophylline as described in FIG. 2.

FIG. 5. Modularity and specificity of the strand displacement-basedribozyme switches. (A) Modular design strategies for the construction ofnew ribozyme switches. The theophylline (left dashed box) andtetracycline (right dashed box) aptamers are shown. (B) Regulatoryactivities of the modular ribozyme switch pair, L2bulge1 and L2bulge1tc,in response to their respective ligands, theophylline (theo) andtetracycline (tc), and closely-related analogues, caffeine (caff) anddoxycycline (doxy). Regulatory effects are reported in fold inductionrelative to the expression levels in the absence of effector asdescribed in FIG. 2.

FIG. 6. System modularity of ribozyme switches enables implementation indiverse cellular engineering applications. (A) System design forribozyme switch-based regulation of cell growth. Small molecule-mediatedregulation of a gene required for cell growth is illustrated for astrand displacement-based ‘OFF’ switch. (B) Theophylline-mediatedribozyme switch-based regulation of cell growth. Changes in growth arereported as OD₆₀₀ values for cells grown in 5 mM 3AT in media lackinghistidine. (C) System design for ribozyme switch-based in vivo sensingof metabolite production. Xanthine is synthesized from fed xanthosineand product accumulation over time is detected through a stranddisplacement-based xanthine-responsive ‘ON’ switch coupled to theregulation of a reporter protein. (D) Ribozyme switch-based xanthinesynthesis detection through L2bulge9. Metabolite sensing throughL2bulge9 is reported in fold induction of GFP levels relative to theexpression levels in the absence of xanthosine feeding as described inFIG. 2. Expression data for experiments performed with L2bulge 1 exhibitsimilar induction profiles and levels (data not shown).

FIG. 7. Control constructs supporting the design strategy forengineering ligand-regulated ribozyme switches. (A) The arrow indicatesthe cleavage site. Sequences of the ribozyme (sTRSV Contl) and stemintegration (hhRz I) controls. (B) Sequences of the loop sequencecontrols in which the loop I and II sequences are replaced by thetheophylline aptamer (L1R and L2R, respectively). (C) Sequences of theloop sequence controls in which the theophylline aptamer is connecteddirectly to the loop I nucleotides through L1.3 and L1.4 (L1Theo) andthe loop II nucleotides through L2.2 and L2.3 (L2Theo). (D) Geneexpression levels (in fold) of the control constructs. 1-fold is definedas the reporter gene expression level of sTRSV relative to that of thebackground fluorescence level. The mean+s.d. from at least threeindependent experiments is shown.

FIG. 8. Flow cytometry histograms of L2bulge1, L2bulgeOff1, and theribozyme control cell populations grown in the presence (+) and absence(−) of 5 mM theophylline. Dark gray line: cell populations grown in theabsence of theophylline; light gray line: cell populations grown in 5 mMtheophylline; shaded population: cell populations indicative of thenon-induced cell population, shaded here to indicate the portion ofcells in the population that have lost the plasmid and exhibitnon-induced, or background, levels of autofluorescence. Histograms arerepresentative of three independent experiments.

FIG. 9. Flow cytometry histograms of the helix slipping-based ribozymeswitch cell populations grown in the presence (+) and absence (−) of 5mM theophylline. Population data is measured and reported as describedin FIG. 8. Histograms are representative of three independentexperiments.

FIG. 10. Flow cytometry histograms of the tuned ribozyme switch seriescell populations grown in the presence (+) and absence (−) of 5 mMtheophylline. Population data is measured and reported as described inFIG. 8. Histograms are representative of three independent experiments.

FIG. 11. Regulatory properties of the helix slipping informationtransmission mechanism. The theophylline-dependent gene regulatorybehavior of L2 cm4 and L1cm10. Gene expression levels are reported asdescribed in FIG. 2, except that L1 Theo is used as a non-switch controlfor L1cm10.

FIG. 12. Temporal responses of L2bulge1, L1cm10, and L2 cm4 in responseto the addition of 5 mM theophylline (final concentration). The timepoint at which theophylline was added to the cultures is indicated by anarrow. Squares: 5 mM theophylline added to growing cultures; diamonds:no theophylline added to growing cultures. Gene expression levels arereported as RFU/OD by dividing fluorescence units by the OD₆₀₀ of thecell sample and subtracting the background fluorescence level. L2bulge1exhibits up-regulation of GFP levels in response to the addition oftheophylline; L1cm10 and L2 cm4 exhibit down-regulation of GFP levels inresponse to theophylline addition. The mean±s.d. from at least threeindependent experiments is shown for all graphs.

FIG. 13. Sequences and structures of tuned ribozyme switches in theL2bulge and L2bulgeOff series'. The nucleotides altered from the parentconstructs, L2bulge1 and L2bulgeOff1, are highlighted. The two stableequilibrium conformations, ribozyme active and inactive conformations,are indicated for the parent ribozyme switches. The ribozyme activeconformations of L2bulge2-5 are not shown as they are similar toL2bulge1. L2bulge6 and L2bulge7 assume a single predominantconformation, ribozyme inactive and ribozyme active, respectively, anddo not undergo theophylline-induced conformational switching. L2bulge8and L2bulge9, modified from L2bulge7 by reducing the stability of theribozyme active conformation and the energy difference between the twoconformations of L2bulge7, now become capable of switching. For thesetwo modified switch constructs, only the ribozyme active conformationsare shown, as their ribozyme inactive conformations are similar to thoseof the other switches illustrated. The ribozyme inactive conformationsof L2bulgeOff2-3 are not shown as they are similar to L2bulgeOff 1.

FIG. 14. Dynamic regulatory ranges of the ribozyme switches and controlsengineered in this work. The regulatory effects at 5 mM theophylline arereported on a full transcriptional range spectrum scale withoutnormalization to the corresponding base expression level of each switchin the absence of effector (0 mM). Little or no effector-mediated generegulatory effect is observed in the non-switch control constructs. Geneexpression fold is defined as previously where 1 fold is equivalent tothe reporter gene expression level of sTRSV relative to the backgroundfluorescence level. sTRSV is the most active ribozyme constructexhibiting the lowest gene expression level and sTRSV Contl is the mostinactive ribozyme construct exhibiting the highest gene expressionlevel, providing a 50 fold range as the full spectrum. Arrows indicatethe direction of regulation as an increasing concentration oftheophylline. These switches offer diverse dynamic ranges of regulationand thus provide a broader utility to fit specific applications ofinterest. Data are reported from three independent experiments.

FIG. 15. Demonstration of theophylline-regulated cell growth by ribozymeswitches through plate-based assays. Cells harboring ribozyme switchesand control constructs were streaked on two plates containing the samemedium except different effector concentrations (0 mM versus 5 mMtheophylline). OFF switches (L1cm10, L2 cm4, L2 cm1, L2bulgeOff1)exhibit suppressed cell growth on the plate containing 5 mM theophyllinewhile an ON switch (L2bulge8) exhibits a higher growth level on theplate containing 5 mM theophylline. The control constructs (L1Theo,L2Theo, sTRSV Contl, and sTRSV) exhibit similar growth levels on bothplates. sTRSV exhibits no cell growth due to its efficient cleavageactivity and sTRSV Contl exhibits the highest levels of growth due toits lack of cleavage activity.

FIG. 16. Detection of intracellular accumulation of the substratexanthosine and the product xanthine over three different time points.Accumulation of xanthosine is observed at earlier time points.Conversion of xanthosine to xanthine was detected at 24 h aftersubstrate feeding and a higher accumulation of xanthine was detected at48 h after substrate feeding.

FIG. 17. Ligand-regulated gene control of the ribozyme switch platformin mammalian cells, demonstrating platform portability across differentcellular systems. All switches exhibit higher gene expression levels inthe presence of 1 mM theophylline compared to those in the absence oftheophylline, while the ribozyme control constructs show littledifference in expression levels in the absence and presence oftheophylline. (#x) indicates the number of independent ribozyme switchesplaced in the 3′ UTR of the target transcript (see next section).

FIG. 18. Ligand-regulated gene regulatory responses and dynamic rangesof single- and multi-copy ribozyme switch constructs. Arrows indicatethe direction of regulation. (#x) indicates the number of independentribozyme switches placed in the 3′ UTR of the target transcript

FIG. 19. The hammerhead ribozyme. A) The catalytic core, NUX sequence,flanking helical regions and Stem II are labeled in the figure. Ovalsrepresent stem loops. B) Ribozyme cleavage mechanism at the NUX triplet.C) Folding of the ribozyme is thought to proceed through two magnesiumbinding events. D) The “Y”-shaped ribozyme is thought to be stabilizedby loop interactions between unpaired bases in the stem loops.

FIG. 20. The anatomy of a thRz with target transcript. The catalyticcore is labeled in the figure. At low Mg²⁺ concentrations, the bulge instem I and stem loop II, interact to stabilize the active conformation.These interactions are found in natural chRzs and have been adapted forengineered thRzs.

FIG. 21. Various thRz designs. A) Previous in vitro studies focused onincorporating the PLMVd-derived stem loops into thRz targeted a segmentof HIV 1 mRNA, PLMVd-L1 thRz. B) By redesigning the targeting arms, weconstructed a series of PLMVd-derived thRzs targeting a sequence inyEGFP.

FIG. 22. Initial ribozyme designs. A) In vivo fluorescence levels of S.cerevisiae strains expressing yEGFP and various trans-ribozyme controlsand constructs. pCS933 is the plasmid only control in both yEGFP(negative control) and no yEGFP (positive control). SSACR represents anoff-target control in which the ribozyme core is maintained while thetargeting arms are scrambled. SSCR1 represents an antisense control inwhich the targeting arms are maintained and the catalytic core isscrambled. SS1 represents a mRz control. B) In vitro cleavage assaysafter 1 hr at various Mg²⁺ concentrations. Target represents the targettranscript alone without the ribozymes added. All other samples aredenoted by the ribozyme added to a reaction mix with target.

FIG. 23. Redesigned expression system. The chRz processing cassette wascloned into pCS933 through AvrII and SacII sites to construct pCS975.thRbz were subsequently cloned between SphI and SacI. This constructexpresses the trans-ribozyme with flanking cis-ribozymes (dashed boxes)on either side of the trans-ribozyme transcript designed to trimtranscript tails, removing the 5′ cap and poly-A tail.

FIG. 24. Initial ribozyme designs with chRz processing cassette. A) Invivo fluorescence levels of S. cerevisiae strains expressing yEGFP andvarious trans-ribozyme controls and constructs. B) In vitro cleavageassays after 1 hr at various Mg²⁺ concentrations.

FIG. 25. Ribozymes with extended targeting arms in chRz processingcassette. In vitro cleavage assays after 1 hr at various Mg²⁺concentrations. At intracellular Mg²⁺ concentrations after 1 hr, thethRz designs with extended arms have processed 80-90% more target thanthe previous thRzs.

FIG. 26. In vitro time points for thRzs with extended targeting arms inchRz processing cassette are fast and highly efficient compared toprevious designs. In vitro cleavage assays after 10, 20, 30, and 60 minat 37° C. and 500 μM MgCl₂.

FIG. 27. Ribozymes with extended targeting arms in chRz processingcassette. In vivo fluorescence levels of S. cerevisiae strainsexpressing yEGFP and various trans-ribozyme controls and constructs. ThethRz designs with extended arms (1154, 1154+5, 1654) exhibit greaterinhibition of expression than the earlier designs.

DETAILED DESCRIPTION OF THE INVENTION

I. Overview

Engineered biological systems hold promise in addressing pressing humanneeds in chemical processing, energy production, materials construction,and maintenance and enhancement of human health and the environment.However, significant advancements in our ability to engineer biologicalsystems have been limited by the foundational tools available forreporting on, responding to, and controlling intracellular components inliving systems. Portable and scalable platforms are needed for thereliable construction of such communication and control systems acrossdiverse organisms.

The present invention provides an extensible RNA-based framework forengineering ligand-controlled gene regulatory systems, called ribozymeswitches, that exhibit tunable regulation, design modularity, and targetspecificity. These switch platforms contain a sensor domain, comprisedof an aptamer sequence, and an actuator domain, comprised of ahammerhead ribozyme sequence. As described in further detail below, theaptamer is directly coupled to a loop of the hammerhead ribozyme. Inaddition, a variety of modes of standardized information transmissionbetween these domains can be employed, and this application demonstratesa mechanism that allows for the reliable and modular assembly offunctioning synthetic hammerhead ribozyme switches and regulation ofribozyme activity in response to various effectors. In addition todemonstrating the first examples of small molecule-responsive, in vivofunctional allosteric hammerhead ribozymes, this application describes ageneral approach for the construction of portable and scalable generegulatory systems. The versatility of the platform in implementingapplication-specific control systems for small molecule-mediatedregulation of cell growth and non-invasive in vivo sensing of metaboliteproduction is described.

This application describes a framework for the reliable de novoconstruction of modular, portable, and scalable control systems that canbe used to achieve flexible regulatory properties, such as up- anddown-regulation of target expression levels and tuning of regulatoryresponse to fit application-specific performance requirements, therebyexpanding the utility of our platforms to a broader range ofapplications. For example, these switch platforms may be applied to theconstruction of transgenic regulatory control systems that areresponsive to cell permeable, exogenous molecules of interest for agiven network. In regulating sets of functional proteins, these switchescan act to rewire information flow through cellular networks andreprogram cellular behavior in response to changes in the cellularenvironment. In regulating reporter proteins, ribozyme switches canserve as synthetic cellular sensors to monitor temporal and spatialfluctuations in the levels of diverse input molecules. The switchplatforms described here represent powerful tools for constructingligand-controlled gene regulatory systems tailored to respond tospecific effector molecules and enable regulation of target genes invarious living systems. Due to their general applicability, ourplatforms offer broad utility for applications in synthetic biology,biotechnology, and health and medicine.

II. Definitions

Hammerhead Ribozyme: A hammerhead ribozyme contains a core, three stemsthat extend from the core, referred to herein as stem I, stem II, andstem III, and at least one loop, which is located on the opposite end ofa stems from the core. In embodiments where the ribozyme is atrans-acting ribozyme, it contains one loop, e.g., at the end of stemII, and is referred to as loop II. In embodiments of cis-actingribozymes, the ribozyme contains two loops, one located at the end ofstem I and is referred to as loop I, and the other located at the end ofstem II and is referred to as loop II.

As used herein, a “cis-cleaving hammerhead ribozyme” is a hammerheadribozyme that, prior to cleavage, is comprised of a singlepolynucleotide. A cis-cleaving hammerhead ribozyme is capable ofcleaving itself.

As used herein, a “trans-cleaving hammerhead ribozyme” is a hammerheadribozyme that, prior to cleavage, is comprised of at least twopolynucleotides. One of the polynucleotides is the target sequence thatis cleaved.

Complementary: Complementary refers to a nucleotide or nucleotidesequence that hybridizes to a given nucleotide or nucleotide sequence.For instance, for DNA, the nucleotide A is complementary to T and viceversa, and the nucleotide C is complementary to G and vice versa. Forinstance, in RNA, the nucleotide A is complementary to the nucleotide Uand vice versa, and the nucleotide C is complementary to the nucleotideG and vice versa. Complementary nucleotides include those that undergoWatson and Crick base pairing and those that base pair in alternativemodes. For instance, as used herein for RNA, the nucleotide G iscomplementary to the nucleotide U and vice versa, and the nucleotide Ais complementary to the nucleotide G and vice versa. Therefore, in anRNA molecule, the complementary base pairs are A and U, G and C, G andU, and A and G. Other combinations, e.g., A and C or C and U, areconsidered to be non-complementary base pairs.

A complementary sequence is comprised of individual nucleotides that arecomplementary to the individual nucleotides of a given sequence, wherethe complementary nucleotides are ordered such that they will pairsequentially with the nucleotides of the given sequence. Such acomplementary sequence is said to be the “complement” of the givensequence. For example, complements of the given sequence, 5′-ACUAGUC-3′,include 3′-UGAUCAG-5′ and 3′-UGGACGG-3′, among others. In the lattersequence, the third and sixth base pairs are both non-Watson and CrickG/U complementary base pairs.

Stem: A stem is a nucleic acid motif that extends from the ribozymecore, at least a portion of which is double-stranded. In certainembodiments, there is a loop at the opposite end of the stem from theribozyme core, and this loop connects the two strands of thedouble-stranded stem. In certain embodiments, a stem comprises 2 to 20complementary base pairs. In certain embodiments, a stem comprises 3, 4,5, 6, 7, 8, or 9 complementary base pairs.

Stems are numbered according to where they extend from the coresequence. In certain embodiments, a hammerhead ribozyme contains threestems, which are referred to as stem I stem II, and stem III. In certainembodiments, stem I extends from the core between the sequence UCH andthe sequence WYGANGA. In certain embodiments, stem II extends from thecore between the sequence WYGANGA and the sequence GAAA. In certainembodiments, stem III extends from the core between the sequence GAAAand the sequence UCH. Thus, in certain embodiments, a ribozyme may beconfigured as follows: 5′-[first strand of stem III] UCH [first strandof stem I] . . . [second strand of stem I] WYGANGA [first strand of stemII] . . . [second strand of stem II] GAAA [second strand of stemIII]-3′. The ellipses in this example represent loop sequences thatconnect the first and second strands of stem I and stem II.

In certain embodiments, at least 30% of the nucleotides in a stem arepart of a complementary base pair. The remaining base pairs may bemismatched, non-complementary base pairs, or may be part of a bulge. Incertain embodiments, at least 40% of the nucleotides in a stem are partof a complementary base pair. In certain embodiments, at least 50% ofthe nucleotides in a stem are part of a complementary base pair. Incertain embodiments, at least 60% of the nucleotides in a stem are partof a complementary base pair. In certain embodiments, at least 70% ofthe nucleotides in a stem are part of a complementary base pair. Incertain embodiments, at least 80% of the nucleotides in a stem are partof a complementary base pair. In certain embodiments, at least 90% ofthe nucleotides in a stem are part of a complementary base pair. Incertain embodiments, at least 95% of the nucleotides in a stem are partof a complementary base pair. In certain embodiments, at least 99% ofthe nucleotides in a stem are part of a complementary base pair. Incertain embodiments, 100% of the nucleotides in a stem are part of acomplementary base pair.

Loop: A loop is a sequence of nucleotides that is not paired withanother strand and is located at the distal end of a stem that isopposite the core. In certain embodiments, a loop is between 1 to 20nucleotides long. In certain embodiments, a loop is between 2 and 10nucleotides long. In certain embodiments, a loop is between 3 and 8nucleotides long. The loop is numbered according to the stem to which itis attached. Therefore, loop I is located at the end of stem I oppositethe core, loop II is located at the end of stem II opposite the core,and loop III is located at the end of stem III opposite the core.

As used herein, a “stem/loop” refers to the entire stem, along with anybulges within that stem, and the loop at the end of the stem. Forexample, stem/loop I includes stem I, including any bulges within stemI, and loop I. If a stem lacks a loop, then stem/loop refers to thestem, along with any bulges within that stem.

Bulge: As used herein, a “bulge” is a sequence of nucleotides that isnot paired with another strand and is flanked on both sides bydouble-stranded nucleic acid sequences. In certain embodiments, a bulgeis located within a stem. When a bulge is located within a stem, thenucleotides of the bulge are considered to be part of the stem. Incertain embodiments, a hammerhead ribozyme comprises more than onebulge. In certain embodiments, a bulge within a stem is located two basepairs from the core. In certain embodiments, one or both strands of thestem contain a bulge.

Directly Coupled: As used herein, an information transmission domain isdirectly coupled to a loop of a ribozyme where the loop, relative toactive ribozyme structure in the absence of the aptamer, is interruptedat one only backbone phosphodiester bond between two residues of theloop, the backbone phosphodiester bond being replaced withphosphodiester bonds to the 5′ and 3′ ends of the aptamer. In the activeform of the aptamer-regulated ribozyme, the 5′ and 3′ residues of theinformation transmission domain are based paired to one another to forma duplex region in order to preserve the structure of the otherwiseinterrupted loop.

Nucleotide: Refers to naturally- and non-naturally-occurring nucleotidesand nucleotide analogs. Nucleotides include, but are not limited to,adenosine, cytosine, guanosine, thymidine, uracil, 4-acetylcytosine,8-hydroxy-N-6-methyladenosine, aziridinyl-cytosine, pseudoisocytosine,5-(carboxyhydroxylmethyl) uracil, 5-fluorouracil, 5-bromouracil,5-carboxymethylaminomethyl-2-thiouracil,5-carboxy-methylaminomethyluracil, dihydrouracil, inosine,N6-iso-pentenyladenine, 1-methyladenine, 1-methylpseudouracil,1-methylguanine, 1-methylinosine, 2,2-dimethyl-guanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-methyladenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyamino-methyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarbonyl-methyluracil, 5-methoxyuracil,2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid methylester,uracil-5-oxyacetic acid, oxybutoxosine, pseudouracil, queosine,2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil,5-methyluracil, N-uracil-5-oxyacetic acid methylester,uracil-5-oxyacetic acid, pseudouracil, queosine, 2-thiocytosine and2,6-diaminopurine.

Nucleic Acid: “nucleic acid sequence,” “nucleic acid molecule,” and“polynucleotide” refer to a DNA sequence or analog thereof, or an RNAsequence or analog thereof. Nucleic acids are formed from nucleotides,including, but not limited to, the nucleotides listed above.

Low Mg²⁺: Refers to a concentration of less than about 1 mM. In certainembodiments, the Mg²⁺ concentration is less than about 0.5 mM Mg²⁺. Incertain embodiments, the Mg²⁺ concentration is less than about 0.1 mMMg²⁺.

Actuator Domain: A switch domain that encodes the system controlfunction. As used here, the actuator domain encodes the gene regulatoryfunction and is comprised of a hammerhead ribozyme sequence.

Communication Module: A sequence element that typically forms animperfectly paired double-stranded stem that can adopt different basepairs between nucleotides through a ‘slip-structure’ mechanism. As usedhere, a communication module is a type of information transmissiondomain that transmits the binding state of the aptamer domain to theadjacent actuator domain through a helix slipping mechanism. Asdemonstrated in this work, a communication module does not act in amodular fashion with other switch domains. The term is retained herefrom earlier work in the field of nucleic acid engineering.

Competing Strand: The nucleic acid sequence within a strand displacementdomain that is bound to the general transmission region of the switchwhen the sensor domain is in the restored conformation (i.e., in thepresence of ligand). The competing strand competes for binding with theswitching strand, which is initially bound to this transmission regionin the absence of ligand.

Component: A part of a system that encodes a distinct activity orfunction.

Composability: A property of a system that indicates its ability to becomprised of components that can be selected and assembled in a modularfashion to achieve a desired system performance. As used here,composability refers to the ability of the individual domains of thecontrol system to be modularly linked without disrupting theiractivities.

Helix Slipping Domain: A subset of information transmission domains thatact through a helix slipping mechanism. The helix slipping domain isalso referred to as the communication module.

Helix Slipping Mechanism: An information transmission mechanism that isbased on an information transmission domain that functions through ahelix slipping event and does not allow for rational design. Such ahelix slipping event utilizes a communication module (or helix slippingdomain) within the general transmission region of the switch (the basestem of the aptamer) to result in disruption or restoration of theactuator domain in response to restoration of the sensor domain.

Information Transmission Domain: A switch domain that encodes thefunction of transmitting information between the sensor domain and theactuator domain.

Information Transmission Mechanism: A general mechanism for transmittinginformation between the sensor domain and the actuator domain of aswitch. As used here, this mechanism regulates the activity of theactuator domain in response to the binding state of the sensor domain.

Ligand: “Ligand” or “analyte” or grammatical equivalents herein is meantto refer to any molecule or compound to be detected and that caninteract with an aptamer to be designed and/or selected as describedhere. Suitable ligands or analytes include, but are not limited to,small chemical molecules such as environmental or clinical chemicals,pollutants or biomolecules, including, but not limited to, pesticides,insecticides, toxins, therapeutic and abused drugs, hormones,antibiotics, antibodies, organic materials, etc. Suitable biomoleculesinclude, but are not limited to, proteins (including enzymes,immunoglobulins and glycoproteins), nucleic acids, lipids, lectins,carbohydrates, hormones, whole cells (including prokaryotic (such aspathogenic bacteria) and eukaryotic cells, including mammalian tumorcells), viruses, spores, etc. Illustrative analytes that are proteinsinclude, but are not limited to, enzymes; drugs; cells; antibodies;antigens; cellular membrane antigens and receptors (neural, hormonal,nutrient, and cell surface receptors) or their natural ligands.

Modular: A property of a system comprised of modules, which indicatesthat the modules can by interchanged as parts without changing theinterface between modules or the modules themselves.

Portability: A property of a system that indicates its ability to beimplemented in environments different from that which it was originallydesigned. As used here, portability refers to the ability of the controlsystem to be implemented in different organisms.

Scalability: A property of a system that indicates its ability to handleincreasing work. As used here, scalability refers to the ability of thecontrol system to be implemented across broad application space by beingable to forward design its response to different molecular information.

Switch: A molecule that can adopt at least two different conformationalstates, where each state is associated with a different activity of themolecule. Often a ligand can bind to one or more conformations of theswitch, such that the presence of the ligand shifts the equilibriumdistribution across the adoptable conformations and therefore regulatesthe activity of the switch molecule. As used here, switch refers to anRNA molecule that can adopt different structures that correspond todifferent gene regulatory activities. An RNA switch is then aligand-controlled gene regulatory system.

Switch Domain: A component of a switch that encodes a distinct activityor function.

Switching Strand: The nucleic acid sequence within a strand displacementdomain that is bound to the general transmission region of the switchwhen the sensor domain is in the disrupted conformation (i.e., in theabsence of ligand). The switching strand is displaced by the competingstrand in the presence of ligand.

Sensor Domain: A switch domain that encodes a ligand binding function.As used here, the sensor domain is comprised of an RNA aptamer sequence.

Strand Displacement Domain: A subset of information transmission domainsthat act through a strand displacement mechanism.

Strand Displacement Mechanism: An information transmission mechanismthat is based on the rational design of an information transmissiondomain that functions through a strand displacement event. Such a stranddisplacement event utilizes competitive binding of two nucleic acidsequences (the competing strand and the switching strand) to a generaltransmission region of the switch (the base stem of the aptamer) toresult in disruption or restoration of the actuator domain in responseto restoration of the sensor domain.

III. Exemplary Embodiments

The hammerhead ribozyme (hRz) is an RNA motif which is capable ofsustaining either in trans or in cis cleavage of a phosphodiester bond.The cis-acting hammerhead ribozyme (chRz) is a catalytic RNA thatundergoes self-cleavage of its own backbone to produce two RNA products.Cis-acting hammerhead ribozymes contain three base-paired stems and ahighly conserved core of residues required for cleavage. The cleavagereaction proceeds by an attack of a 2′ hydroxyl oxygen of a catalyticsite cytosine on the phosphorus atom attached to the 3′ carbon of thesame residue. This breaks the sugar phosphate backbone and produces a2′,3′ cyclic phosphate.

The minimal hammerhead sequence that is required for the self-cleavagereaction includes approximately 13 conserved or invariant “core”nucleotides, most of which are not involved in forming canonicalWatson-Crick base-pairs. The core region is flanked by stems I, II andIII, which are in general comprised of canonical Watson-Crick base-pairsbut are otherwise not constrained with respect to sequence.

Cleavage specificity of the trans-acting hammerhead ribozyme (thRz) iscontrolled by the hybridizing arms of the ribozyme, which anneal withthe substrate in a complementary fashion and direct cleavage of thescissile phosphodiester bond. This activity is specifically directed tooccur after the third nucleotide of the cleavage triplet.

The present invention provides aptamer-regulated trans-acting hammerheadribozymes and aptamer-regulated cis-acting hammerhead ribozymes. Thesubject aptamer-regulated thRzs and chRzs are a versatile class ofribozymes that can be readily engineered to be responsive to a varietyof ligands, and are useful in many applications. For example,aptamer-regulated thRzs and chRzs can be designed to modulate theactivity of targeted genes in a ligand-dependent manner, and aretherefore useful for modulating the expression of endogenous orheterologous genes.

The ribozyme domain (also herein the effector domain) can have at leasttwo conformational states, an “off” state and an “on” state, that isdefined by its activity level (reaction rate, for example) for eitherundergoing self-cleavage in the case of chRzs, or cleaving a targetsequence in the case of thRzs. The effector domains of the invention canbe switched between their “on” and “off” conformational states inresponse to ligand binding to the aptamer domain. Aptamer-regulatedribozymes of the invention, therefore, act as a switch whose activity isturned “on” and “off” in response to ligand binding. In certainembodiments, the ribozyme domain's function is starkly dependent on thepresence or absence of the ligand, or can show a more dose-response likedependency on concentration of the ligand available to bind to theaptamer domain.

The choice of ligand to which the aptamer binds, and the ribozymetherefore is regulated by, are vast. In certain instances, the ligand isa small molecule having a molecular weight less than 2500 amu.

These can be naturally or non-naturally occurring molecules, includingpeptides, small organic molecules (including drugs and certainmetabolites and intermediates, cofactors, etc), and metal ions merely toillustrate. Exemplary ligands that bind to an aptamer include, withoutlimitation, small molecules, such as drugs, metabolites, intermediates,cofactors, transition state analogs, ions, metals, nucleic acids, andtoxins. Aptamers may also bind natural and synthetic polymers, includingproteins, peptides, nucleic acids, polysaccharides, glycoproteins,hormones, receptors and cell surfaces such as cell walls and cellmembranes. The binding of a ligand to an aptamer, which is typicallyRNA, alters the base-pairing with the information transmission domainthat is carried over as a structural change in the ribozyme domain andalters its ability to mediate cleavage of a phosphodiester bond (eitherself-cleavage or cleavage of a target sequence). Therefore, ligandbinding affects the effector domain's ability to mediate geneinactivation, transcription, translation, or otherwise interfere withthe normal activity of the target gene or mRNA, for example. An aptamerwill most typically have been obtained by in vitro selection for bindingof a target molecule. However, in vivo selection of an aptamer is alsopossible. Aptamers have specific binding regions which are capable offorming complexes with an intended target molecule in an environmentwherein other substances in the same environment are not complexed tothe nucleic acid. The specificity of the binding is defined in terms ofthe comparative dissociation constants (K_(d)) of the aptamer for itsligand as compared to the dissociation constant of the aptamer for othermaterials in the environment or unrelated molecules in general. A ligandis one which binds to the aptamer with greater affinity than tounrelated material. Typically, the K_(d) for the aptamer with respect toits ligand will be at least about 10-fold less than the K_(d) for theaptamer with unrelated material or accompanying material in theenvironment. Even more preferably, the K_(d) will be at least about50-fold less, more preferably at least about 100-fold less, and mostpreferably at least about 200-fold less. An aptamer will typically bebetween about 10 and about 300 nucleotides in length. More commonly, anaptamer will be between about 30 and about 100 nucleotides in length.

Aptamers are readily made that bind to a wide variety of molecules. Eachof these molecules can be used as a modulator of the associated ribozymeusing the methods of the invention. For example, organic molecules,nucleotides, amino acids, polypeptides, target features on cellsurfaces, ions, metals, salts, saccharides, have all been shown to besuitable for isolating aptamers that can specifically bind to therespective ligand. For instance, organic dyes such as Hoechst 33258 havebeen successfully used as target ligands for in vitro aptamer selections(Werstuck and Green, Science 282:296-298 (1998)). Other small organicmolecules like dopamine, theophylline, sulforhodamine B, and cellobiosehave also been used as ligands in the isolation of aptamers. Aptamershave also been isolated for antibiotics such as kanamycin A,lividomycin, tobramycin, neomycin B, viomycin, chloramphenicol andstreptomycin. For a review of aptamers that recognize small molecules,see Famulok, Science 9:324-9 (1999).

In certain embodiments, the ligand of the aptamer of anaptamer-regulated ribozyme of the invention is a cell-permeable, smallorganic molecule. Small organic molecules which do not have a generalinhibitory effect on translation are preferred as ligands. The smallmolecule preferably also exhibits in vivo persistence sufficient forachieving the desired level of inhibition of translation. The moleculesalso can be screened to identify those that are bioavailable after, forexample, oral administration. In certain embodiments of the invention,the ligand is nontoxic. The ligand may optionally be a drug, including,for example, a steroid. However, in some of the methods of controllinggene expression, it is preferable that the ligand be pharmacologicallyinert. In some embodiments, the ligand is a polypeptide whose presencein the cell is indicative of a disease or pathological condition. Inother embodiments, the ligand for an aptamer is an antibiotic, such aschloramphenicol. In an alternative embodiment, the ligand of the aptameris an organic dye such as Hoeschst dye 33258. In still anotherembodiment, the ligand may be a metal ion. In a specific embodiment, theaptamer domain of an aptamer-regulated nucleic acid responds to bindingto caffeine.

Aptamers are typically developed to bind particular ligands by employingknown in vivo or in vitro (most typically, in vitro) selectiontechniques known as SELEX (Ellington et al., Nature 346, 818-22 (1990);and Tuerk et al., Science 249, 505-10 (1990)). Methods of makingaptamers are also described in, for example, U.S. Pat. No. 5,582,981,PCT Publication No. WO 00/20040, U.S. Pat. No. 5,270,163, Lorsch andSzostak, Biochemistry, 33:973 (1994), Mannironi et al., Biochemistry36:9726 (1997), Blind, Proc. Nat'l. Acad. Sci. USA 96:3606-3610 (1999),Huizenga and Szostak, Biochemistry, 34:656-665 (1995), PCT PublicationNos. WO 99/54506, WO 99/27133, WO 97/42317 and U.S. Pat. No. 5,756,291.

Generally, in their most basic form, in vitro selection techniques foridentifying aptamers involve first preparing a large pool ofoligonucleotides of the desired length that contain at least some regionthat is randomized or mutagenized. For instance, a commonoligonucleotide pool for aptamer selection might contain a region of20-100 randomized nucleotides flanked on both ends by an about 15-25nucleotide long region of defined sequence useful for the binding of PCRprimers. The oligonucleotide pool is amplified using standard PCRtechniques, although any means that will allow faithful, efficientamplification of selected nucleic acid sequences can be employed. TheDNA pool is then in vitro transcribed to produce RNA transcripts. TheRNA transcripts may then be subjected to affinity chromatography,although any protocol which will allow selection of nucleic acids basedon their ability to bind specifically to another molecule (e.g., aprotein or any target molecule) may be used. In the case of affinitychromatography, the transcripts are most typically passed through acolumn or contacted with magnetic beads or the like on which the targetligand has been immobilized. RNA molecules in the pool which bind to theligand are retained on the column or bead, while nonbinding sequencesare washed away. The RNA molecules which bind the ligand are thenreverse transcribed and amplified again by PCR (usually after elution).The selected pool sequences are then put through another round of thesame type of selection. Typically, the pool sequences are put through atotal of about three to ten iterative rounds of the selection procedure.The cDNA is then amplified, cloned, and sequenced using standardprocedures to identify the sequence of the RNA molecules which arecapable of acting as aptamers for the target ligand. Once an aptamersequence has been successfully identified, the aptamer may be furtheroptimized by performing additional rounds of selection starting from apool of oligonucleotides comprising the mutagenized aptamer sequence.For use in the present invention, the aptamer is preferably selected forligand binding in the presence of salt concentrations and temperatureswhich mimic normal physiological conditions.

One can generally choose a suitable ligand without reference to whetheran aptamer is yet available. In most cases, an aptamer can be obtainedwhich binds the ligand of choice by someone of ordinary skill in theart. The unique nature of the in vitro selection process allows for theisolation of a suitable aptamer that binds a desired ligand despite acomplete dearth of prior knowledge as to what type of structure mightbind the desired ligand.

For an aptamer to be suitable for use in the present invention, thebinding affinity of the aptamer for the ligand must be sufficientlystrong and the structure formed by the aptamer when bound to its ligandmust be significant enough so as to switch an aptamer-regulated ribozymeof the invention between “on” and “off” states or tune the functionallevel of an aptamer-regulated ribozyme.

The association constant for the aptamer and associated ligand ispreferably such that the ligand functions to bind to the aptamer andhave the desired effect at the concentration of ligand obtained uponadministration of the ligand. For in vivo use, for example, theassociation constant should be such that binding occurs well below theconcentration of ligand that can be achieved in the serum or othertissue. Preferably, the required ligand concentration for in vivo use isalso below that which could have undesired effects on the organism.

Accordingly, certain embodiments provide methods of designing andselecting aptamers or aptamer domains that are responsive to one or morepre-selected or pre-determined ligands. The subject aptamer-regulatedribozymes may also be “tuned” so that their switching behavior is moreor less responsive to ligand binding. Aptamer-regulated ribozymes mayalso be “tuned” so that the binding affinity of the aptamer domain ismore or less sensitive to its ligand. For instance, the thermodynamicproperties of intramolecular duplex formation and other 2° and 3°structures in the aptamer-regulated ribozymes may be altered so that theaptamer domain is more or less amenable to ligand binding, i.e., such asmay be manifest in the dissociation constant (K_(a)) or other kineticparameters (such as K_(on) and K_(off) rates). Alternatively, allostericchanges in the ribozyme domain may be more or less responsive to ligandbinding upon alterations in hybridization and other intramolecularinteractions that may effect 2° and 3° structures of the ribozymedomain. Forward engineering strategies for altering the thermodynamicproperties of nucleic acid structures are well known in the art. Forinstance, increased complementary nucleic acid pairing may increase thestability of a ribozyme domain or aptamer domain.

EXAMPLE 1 Generation of Modular and Scalable Aptamer-RegulatedCis-Ribozymes

To satisfy the engineering design principle of scalability (DP1) wechose RNA aptamers (11), nucleic acid ligand-binding molecules, as thesensing platform for the universal control system. Our choice of sensingplatform was driven by the proven versatility of RNA aptamers. Standardin vitro selection strategies or SELEX (12, 13) have been used togenerate RNA aptamers de novo to a wide variety of ligands, includingsmall molecules, peptides, and proteins (14). In addition, thespecificity and affinity of an aptamer can be tuned through theselection process to meet the specific performance requirements of agiven application. The continued selection of new aptamers toappropriate cellular molecules that function under in vivo conditionswill enable these elements to be implemented as sensors in RNA-basedcontrol systems.

To satisfy the engineering design principle of portability (DP2) wechose the hammerhead ribozyme, a catalytic RNA, as the regulatoryelement in the universal control system. Our choice of regulatoryelement was driven by the ability of the hammerhead ribozyme to exhibitself-cleavage activity across various organisms and its demonstratedpotential in biomedical and biotechnological applications owing to itssmall size, relative ease of design, and rapid kinetics (15). Theutility of hammerhead ribozymes as gene regulatory elements has beendemonstrated in various systems (16-18). In addition, several researchgroups have engineered a special class of synthetic hammerhead ribozymesreferred to as allosteric hammerhead ribozymes that contain separatecatalytic and ligand-binding domains, which interact in aligand-dependent manner to control the activity of the ribozyme (19-22).While this class of ribozymes enables a better control system due to thepresence of the integrated ligand-binding domain, there has been nosuccess in translating them to in vivo environments.

Design Strategies for Engineering Portability, Utility, andComposability into a Biological Control System

To support a framework for engineering ligand-controlled gene regulatorysystems, we specified a design strategy that is in accordance with ourengineering principles stated above (FIG. 1A, B). This strategy iscomprised of three components that address mechanisms for theportability (DP2), utility (DP3), and composability (DP4) of the controlsystem and are critical to the development of a general ribozyme switchplatform. First, the cis-acting hammerhead ribozyme constructs areintegrated into the flexible regulatory space of the 3′ UTR (FIG. 1A).We chose to locate the synthetic ribozymes within the 3′ UTR of theirtarget gene as opposed to the 5′ UTR in order to isolate their specificcleavage effects on transcript levels from their non-specific structuraleffects on translation initiation, as secondary structures have beendemonstrated to repress efficient translation when placed in the 5′ UTR(23; K. Hawkins and C.D.S., unpublished observations). In addition,cleavage within the 3′ UTR is a universal mechanism for transcriptdestabilization in eukaryotic and prokaryotic organisms. Second, eachribozyme construct is insulated from surrounding sequences, which maydisrupt its structure and therefore its activity, by incorporatingspacer sequences immediately 5′ and 3′ of stem III (FIG. 1A). Byimplementing these two components, we ensure that these control systemswill be portable across organisms and modular to coupling with differentcoding regions. The third component was necessitated by the fact thatprevious engineered in vitro allosteric ribozyme systems, which replacestem loops I or II with part of the aptamer domain (FIG. 1B, lowerright), do not function in vivo. From previous studies on the satelliteRNA of tobacco ringspot virus (sTRSV) hammerhead ribozyme (17), wesuspect that this lack of in vivo functionality in earlier designsresults from removal of stem loop sequences that may play a criticalrole in tertiary interactions that stabilize the catalytically activeconformation under physiological Mg²⁺ concentrations. To developribozyme switches that function in vivo, we chose to integrate thehammerhead ribozyme into the target transcript through stem III andcouple the sensor domain directly to the ribozyme through stem loops Ior II to maintain these potentially essential sequence elements (FIG.1B, upper right). Construction and characterization of the regulatoryactivity of a series of ribozyme control constructs in the eukaryoticmodel organism Saccharomyces cerevisiae (FIG. 1A) indicate thatmaintenance of loop I and II sequences and thus integration through stemIII are essential for their in vivo functionality (Example 2, FIG. 7).

Engineering Mechanisms for Information Transmission Between the ModularSwitch Domains

The final design challenge in building a universal switch platform is todevelop a standardized means of transmitting information (encoded withinan information transmission domain) from the sensor (aptamer) domain tothe regulatory (ribozyme) domain (DP5). There are two differentstrategies for transmitting information between the aptamer and ribozymedomains: strand displacement and helix slipping. We constructed andcharacterized ribozyme switch platforms based on both mechanisms.

The first information transmission domain that we developed is based ona strand displacement mechanism, which involves the rational design oftwo sequences that compete for binding to a general transmission region(the base stem of the aptamer) (FIG. 2A, B). We employed this mechanismin engineering a ribozyme switch platform that enables both up- anddown-regulation of gene expression in response to increasing effectorconcentrations (‘ON’ and ‘OFF’ switches, respectively). An initialribozyme switch, L2bulge1, was constructed to up-regulate geneexpression through the corresponding base platform (L2Theo, FIG. 7C) byincorporating a competing strand following the 3′ end of thetheophylline aptamer (24) (FIG. 2A). This competing strand is perfectlycomplementary to the base stem of the aptamer at the 5′ end. Using thesame design principles, we engineered another ribozyme switch,L2bulgeOff1 (FIG. 2B), for down-regulating gene expression. Our stranddisplacement strategy is based on the conformational dynamicscharacteristic of RNA molecules that enables them to distribute betweenat least two different conformations at equilibrium: one conformation inwhich the competing strand is not base-paired or base-paired such thatthe ligand-binding pocket is not formed, and the other conformation inwhich the competing strand is base-paired with the aptamer base stem,displacing the switching strand and thus allowing the formation of theligand-binding pocket. Strand displacement results in the disruption(L2bulge1) or restoration (L2bulgeOff1) of the ribozyme's catalyticcore. Binding of theophylline to the latter conformation shifts theequilibrium distribution to favor the aptamer-bound form as a functionof increasing theophylline concentration. An increase in targetexpression levels (induction in fold 25) at 5 mM theophylline relativeto those in the absence of effector was observed in L2bulge1 (FIG. 2Cand FIG. 8). In contrast, a reduction in expression levels (reduction infold 18) at 5 mM theophylline relative to those in the absence ofeffector was observed in L2bulgeOff1 (FIG. 2D and FIG. 8). Through ourstrand displacement mechanism, we have engineered ribozyme switches denovo that provide allosteric regulation of gene expression and functionas ‘ON’ and ‘OFF’ switches.

We engineered a second ribozyme switch platform to examine analternative information transmission domain based on a helix slippingmechanism, which does not allow for rational design (FIG. 3A). Thismechanism involves the functional screening of ‘communication modules’(20-22) within the base stem of the aptamer. Communication modules aredynamic elements capable of transmitting the binding state of an aptamerdomain to an adjacent regulatory domain through a ‘slip-structure’mechanism (20), in which a nucleotide shift event is translated to asmall-scale change in the conformation of the regulatory domain in aligand-dependent manner. These elements have been developed through invitro screening processes, and their communicative properties have beendemonstrated in vitro in engineered allosteric ribozymes (19-22). Wescreened the in vivo functionality of previously in vitro selectedcommunication modules (20-22) by assaying the activity of thesesequences within L1 Theo and L2Theo. A critical difference between thedesign of the previously developed in vitro allosteric ribozymes, fromwhich these communication modules were generated, and that of ourengineered ribozyme switches is the coupling strategies between theaptamer and ribozyme domains and their effects on the in vivo activityof the ribozyme domain as described previously (FIG. 1B). Among thethirteen communication modules (20-22) screened for in vivo activity,five (cm1, cm4, cm5, cm9, and cmd) exhibit down-regulation of expressionlevels through loop II, whereas only two (cm10 and cmd) exhibit suchregulation through loop I (FIG. 3B). The regulatory activities of twohelix slipping-based ribozyme switches, L2 cm4 and L1cm10 (FIGS. 9, 11),were characterized across a range of theophylline concentrations andexhibit substantial regulatory effects. Although the helix slippingconstructs are comprised of identical aptamer and catalytic coresequences, they exhibit different extents of regulation. Thisvariability suggests that each construct contains a differentequilibrium distribution between the adoptable conformations and thatthe energy required for structural switching between the conformationsis also different.

We validated the regulatory mechanisms of representative stranddisplacement- and helix slipping-based switches. Relative steady-statetranscript levels in the absence and presence of effector are consistentwith corresponding fluorescent protein levels, indicating that cleavagein the 3′ UTR results in rapid decay and inactivation of the targettranscript. In addition, we demonstrated that changes in expressionlevels are induced shortly after effector addition (FIG. 12), indicatingthat the response of the regulatory elements to changes in effectorlevels is relatively rapid.

Rational Tuning Strategies Enable Programming of Switch RegulatoryResponse

The ability to program the regulatory response of a universal switchplatform is an important property in tuning the platform performance tocomply with the design specifications for a particular application. Wedemonstrate that our strand displacement-based switch platformincorporates an information transmission mechanism that is amenable torational tuning strategies for programming response properties.Programming of new regulatory information is achieved by sequencealteration resulting in a change in the molecule's structural stability,which may affect its switching dynamics if the molecule can adoptmultiple conformations. These rational sequence modification tuningstrategies are not applicable to communication module-based switches dueto an inability to predict their activities. A more complete descriptionof our tuning strategies is provided in Example 2, FIGS. 13-14. Briefly,our rational tuning strategies target alteration of the nucleotidecomposition of the base stem of the aptamer domain to affect thestabilities of individual constructs and the energies required for theconstruct to switch between two adoptable conformations. Using thesestrategies, we rationally engineered a series of tuned ‘ON’ and ‘OFF’switches from L2bulge 1 and L2bulgeOff1, respectively (FIG. 4A). Thesetuned switches exhibit different regulatory ranges in accordance withour rational energetic tuning strategies (FIGS. 4B, C, and FIG. 10).

The Ribozyme Switch Platform Exhibits Component Modularity andSpecificity

In implementing a standardized mechanism through which to transmitinformation between the domains of a switch platform (DP5), we needed toconfirm that the modular coupling between the aptamer and ribozymedomains is maintained (DP4). We performed modularity studies on ourstrand displacement-based ribozyme switch platform, in which aptamerspossessing sequence flexibility in their base stems can be swapped intothe sensor domain. To begin to demonstrate that ribozyme switch activitymay be controlled by different effector molecules we replaced thetheophylline aptamer of L2bulge1 with a tetracycline mini-aptamer (25)to construct a tetracycline-responsive ‘ON’ switch (L2bulge1tc) (FIG.5A). Despite similar aptamer ligand affinities (24, 25), the extent ofup-regulation with L2bulge1tc was greater than that with L2bulge1 at thesame extracellular concentration of their respective ligands (FIG. 5B).This is likely due to the high cell permeability of tetracycline (26)compared to theophylline (27). These results demonstrate that our stranddisplacement-based switch platform maintains modularity between theaptamer and ribozyme domains. We also performed similar modularitystudies on the helix slipping-based switch platform by replacing thetheophylline aptamer of L1cm10, L2 cm4 and L2 cm5 with the tetracyclinemini-aptamer (L1cm10, L2 cm4tc and L2 cm5tc, respectively). Theseconstructs do not exhibit effector-mediated gene regulatory effects(data not shown). We also demonstrated that the aptamer sequences(theophylline and tetracycline) incorporated into our ribozyme switchplatforms maintain highly specific target recognition capabilities invivo similar to their in vitro specificities generated during theselection process against corresponding molecular analogues (caffeineand doxycycline, respectively) (24, 25) (FIG. 5B). This is an importantproperty in implementing these platforms in cellular engineeringapplications that involve complex environments where molecular speciessimilar to the target ligand may be present.

Component Modularity Enables Implementation of Ribozyme Switches asRegulatory Systems in Diverse Applications

To demonstrate the scalability and utility of these switch platforms asapplication-specific control systems, we demonstrate the implementationof ribozyme switches in two distinct cellular engineering applicationareas. First, utility (DP3) and the ability to respond to and controlcellular information is demonstrated by the application of ribozymeswitches to small molecule-mediated regulation of cell growth. Second,scalability (DP1) and the ability to respond to and report on cellularinformation is demonstrated by the implementation of ribozyme switchesas non-invasive in vivo sensors of metabolite production.

The first system explores the application of our ribozyme switches tothe regulation of a survival gene, where modification of expressionlevels is expected to produce an observable and titratable phenotypiceffect on cell growth. The reporter gene within the original constructswas replaced with a growth-associated gene (his5) responsible for thebiosynthesis of histidine in yeast (28) (FIG. 6A). We performed growthregulation assays across various effector concentrations usingrepresentative switch constructs and demonstrated that these switchesmediate cell growth in a highly effector-dependent manner (FIG. 6B andFIG. 15). This application demonstrates the utility (DP3) of our switchplatform, in which the control system exhibits modularity to thefunctional level components in the regulatory system.

The second system explores the application of these ribozyme switches tothe in vivo sensing of metabolite production to demonstrate that theseswitches provide a non-invasive mechanism through which to transmitmolecular information from cells. Nucleoside phosphorylase activitiesresulting in N-riboside cleavage of purine nucleosides have beenidentified in various organisms (29). We observe that feeding xanthosineto our yeast cultures results in the production of xanthine, a productsynthesized through riboside cleavage of xanthosine. The theophyllineaptamer employed in our switch platforms possesses a reduced bindingaffinity for xanthine (27-fold lower than theophylline) (24). Weemployed two ‘ON’ switch constructs (L2bulge1 and L2bulge9) for the invivo detection of xanthine production in cultures fed xanthosine (FIG.6C). GFP levels in cells fed xanthosine rose steadily between 24-40 hpost-feeding in correlation with HPLC data (FIG. 6D and FIG. 16),illustrating the non-invasive metabolite-sensing capabilities of theseswitches through transmitting changes in metabolite accumulation tochanges in reporter expression levels. This application demonstrates thescalability (DP1) of our switch platform, in which the unique propertiesof the sensing platform employed in this control system enable broadimplementation in diverse applications not generally accessible by otherregulatory systems.

Materials and Methods

-   Plasmid and Switch Construction. Using standard molecular biology    techniques (30), a characterization plasmid, pRzS, harboring the    yeast-enhanced green fluorescence protein (yEGFP) (31) under control    of a GAL1-10 promoter, was constructed. For the ribozyme    switch-mediated growth studies, the yegfp gene was replaced with the    his5 gene (28). See Example 2 for details.-   RNA Secondary Structure Prediction and Free Energy Calculation.    RNAstructure 4.2 (on the World Wide Web at    rna.urmc.rochester.edu/rnastructure.html) was used to predict the    secondary structures of all switch constructs and their    thermodynamic properties. RNA sequences that are predicted to adopt    at least two stable equilibrium conformations (ribozyme inactive and    active) were constructed and examined for functional activity.-   Ribozyme Characterization, Cell Growth Regulation, and Metabolite    Sensing Assays. See Example 2 for details. Briefly, cells harboring    appropriate plasmids were grown in the absence and presence of    corresponding ligands or substrates and characterized for    ligand-regulated ribozyme switch activity, cell growth, and    metabolite sensing.-   Fluorescence quantification. See Example 2 for details.-   Quantification of Cellular Transcript Levels. See Example 2 for    details. Briefly, total RNA was extracted employing standard acid    phenol methods (32) followed by cDNA synthesis and PCR    amplification.

REFERENCES CITED IN EXAMPLE 1

-   1. Endy D (2005) Nature 438:449-53.-   2. Voigt C A (2006) Curr Opin Biotechnol 17:548-57.-   3. Kobayashi H, Kaern M, Araki M, Chung K, Gardner T S, Cantor C R,    Collins J J (2004) Proc Natl Acad Sci USA 101:8414-9.-   4. Gossen M, Bujard H (1992) Proc Natl Acad Sci USA 89:5547-51.-   5. Lutz R, Bujard H (1997) Nucleic Acids Res 25:1203-10.-   6. Mandal M, Breaker R R (2004) Nat Rev Mol Cell Biol 5:451-63.-   7. Kim D S, Gusti V, Pillai S G, Gaur R K (2005) RNA 11:1667-77.-   8. An C I, Trinh V B, Yokobayashi Y (2006) RNA 12:710-6.-   9. Bayer T S, Smolke C D (2005) Nat Biotechnol 23:337-43.-   10. Isaacs F J, Dwyer D J, Collins J J (2006) Nat Biotechnol    24:545-54.-   11. Bunka D H, Stockley P G (2006) Nat Rev Microbiol 4:588-96.-   12. Tuerk C, Gold L (1990) Science 249:505-10.-   13. Ellington A D, Szostak J W (1990) Nature 346:818-22.-   14. Hermann T, Patel D J (2000) Science 287:820-5.-   15. Birikh K R, Heaton P A, Eckstein F (1997) Eur J Biochem    245:1-16.-   16. Marschall P, Thomson J B, Eckstein F (1994) Cell Mol Neurobiol    14:523-38.-   17. Khvorova A, Lescoute A, Westhof E, Jayasena S D (2003) Nat    Struct Biol 10:708-12.-   18. Yen L, Svendsen J, Lee J S, Gray J T, Magnier M, Baba T, D'Amato    R J, Mulligan R C (2004) Nature 431:471-6.-   19. Koizumi M, Soukup G A, Kerr J N, Breaker R R (1999) Nat Struct    Biol 6:1062-71.-   20. Soukup G A, Breaker R R (1999) Proc Natl Acad Sci USA 96:3584-9.-   21. Soukup G A, Emilsson G A, Breaker R R (2000) J Mol Biol    298:623-32.-   22. Kertsburg A, Soukup G A (2002) Nucleic Acids Res 30:4599-606.-   23. Pelletier J, Sonenberg N (1985) Cell 40:515-26.-   24. Jenison R D, Gill S C, Pardi A, Polisky B (1994) Science    263:1425-9.-   25. Berens C, Thain A, Schroeder R (2001) Bioorg Med Chem 9:2549-56.-   26. Hanson S, Berthelot K, Fink B, McCarthy J E, Suess B (2003) Mol    Microbiol 49:1627-37.-   27. Koch A L (1956) J Biol Chem 219:181-8.-   28. Nishiwaki K, Hayashi N, Irie S, Chung D H, Harashima S, Oshima    Y (1987) Mol Gen Genet. 208:159-67.-   29. Ogawa J, Takeda S, Xie S X, Hatanaka H, Ashikari T, Amachi T,    Shimizu S (2001) Appl Environ Microbiol 67:1783-7.-   30. Sambrook J, Russell D W (2001) Molecular cloning: a laboratory    manual (Cold Spring Harbor Laboratory Press, Cold Spring Harbor,    N.Y.-   31. Mateus C, Avery S V (2000) Yeast 16:1313-23. 32. Caponigro G,    Muhlrad D, Parker R (1993) Mol Cell Biol 13:5141-8.

EXAMPLE 2 Supplemental Data for Aptamer-Regulated Cis-Ribozymes

Ribozyme Control Constructs for Loop Sequence Coupling and StemIntegration Controls

To establish and make useful our design strategy we constructed andcharacterized a series of ribozyme controls. We characterized theregulatory activity of our ribozyme constructs within a modular ribozymecharacterization system in the eukaryotic model organism Saccharomycescerevisiae (FIG. 1A). First, an inactive ribozyme control (sTRSV Contl)was constructed to adopt the same structural motif as sTRSV (FIG. 1A),while carrying a scrambled catalytic core sequence (FIG. 7A). Second, asynthetic sTRSV ribozyme (hhRz I) that contains closed loops in stems IIand III and is embedded through stem I was constructed as a stemintegration control (FIG. 7A). Finally, we constructed four loopsequence controls. In one set, stem loops I and II (L1R and L2R,respectively) were replaced by the theophylline aptamer TCT8-4 (S1)(FIG. 7B), and in another set, the theophylline aptamer was coupleddirectly to sequences in loops I and II (L1Theo and L2Theo,respectively) (FIG. 7C). sTRSV exhibits a 50-fold reduction in targetexpression levels relative to sTRSV Contl (FIG. 7D). HhRz I, UR, and L2Rexhibit similar target expression levels to that of sTRSV Contl,suggesting that ribozyme activity was abolished in these constructs. Incontrast, L1Theo and L2Theo exhibit significantly lower targetexpression levels relative to sTRSV Contl. L1Theo and L2Theo wereemployed as the primary base constructs in engineering our syntheticribozyme switch platforms. In addition, scrambled core versions ofL1Theo and L2Theo exhibit no theophylline-dependent shifts in geneexpression (data not shown), indicating that theophylline binding inthat region of the transcript alone is not responsible for the observedregulatory effects. Taken together, we find that our design strategyenables the construction of a universal ribozyme switch platform thatsatisfies the design principles of portability, utility, andcomposability.

Rational Tuning Strategies for Strand Displacement-Based Switches

A series of nine tuned ‘ON’ switches were constructed from L2bulge1 as abase structure by employing rational energetic tuning strategiesdeveloped in this work. This strategy is based on the effects ofaltering the predicted free energies of a particular conformation (−ΔG)and the predicted difference between the free energies of twoconformations (ΔΔG) on RNA conformational dynamics, or the ability ofthe RNA molecule to distribute between these two conformational states.Lowering values for either of these energetic measurements (−ΔG or ΔΔG)is expected to make it easier for a particular RNA molecule to switchbetween the conformational states in question. Therefore, there is ananticipated optimum conformational energy and energetic differencebetween conformations to achieve the desired range of switching inresponse to effector concentration (i.e., energy measurements too highwill result in stable non-switch designs, and energy measurements orenergy difference measurements too low will result in fairly equaldistributions between the two conformational states and lower switchingcapabilities). It is also expected, then, that one can “push” switchesinto a non-switch state by moving away from this energetic optimum. Thisstrategy was examined in a series of tuning experiments described below.

L2bulge2 and L2bulge3 (FIG. 13) replace canonical base pairs in theaptamer base stem of the ribozyme inactive conformation of L2bulge1 withU-G wobble pairs. As a result of these destabilizing alterations, bothequilibrium conformations (ribozyme active and ribozyme inactive) becomeless thermodynamically stable than those of L2bulge1, as estimated fromtheir predicted free energies (−ΔG). In addition, the energy required toswitch between the two equilibrium conformations was maintained similarto that of L2bulge1, as estimated by the difference between the freeenergies of the two conformations (ΔΔG). Ribozyme assays indicate thatboth L2bulge2 and L2bulge3 exhibit smaller dynamic ranges than that ofL2bulge1 (FIG. 4B and FIG. 14). It is proposed that the lowerstabilities of the conformational states enable more frequent dynamicswitching between the two equilibrium conformations and therefore lowerthe difference in distribution favoring one state over the other.

L2bulge4 (FIG. 13) incorporates an additional G-U wobble pair within theaptamer base stem of the ribozyme inactive conformation of L2bulge1.However, this aptamer stem extension does not result in an appreciablepredicted change in the thermodynamic stabilities of the equilibriumconformations or the energy required to switch between the twoequilibrium conformations when compared to L2bulge1. Ribozyme assaysindicate that L2bulge4 exhibits a dynamic range in response totheophylline levels similar to that of L2bulge1 (FIG. 4B and FIG. 14).

L2bulge5 (FIG. 13) incorporates an additional canonical base pair (A-U)within the aptamer base stem of L2bulge1. As a result of thisstabilizing alteration, the conformation of the ribozyme switch, inwhich the aptamer structure is formed and the catalytic core isdisrupted (ribozyme inactive), is increased in stability and as stableas the conformation in which the catalytic core is not disrupted(ribozyme active). The increased stability of the ribozyme inactiveconformation in L2bulge5 in comparison to L2bulge1 and L2bulge4indicates that the equilibrium distribution between these twoconformations will shift to favor the ribozyme inactive conformation.Ribozyme assays indicate that L2bulge5 exhibits significantly higher GFPexpression levels in the absence and presence of theophylline comparedto those of L2bulge1 and L2bulge4, such that the theophylline-regulatedincrease in gene expression is similar to that of L2bulge3 but differentin regulatory dynamic ranges (FIG. 4B and FIG. 14).

Two switches in this series, L2bulge6 and L2bulge7, were constructed todemonstrate the ability of this tuning strategy to “push” the ribozymeswitch constructs out of a switchable energetic range and approachnon-switching extremes. L2bulge6 (FIG. 13) was designed to energeticallyfavor the conformation, in which the aptamer structure is formed and thecatalytic core is disrupted, (ribozyme inactive) in the absence oftheophylline by introducing a stabilizing G-C base pair into the aptamerstem of this conformation. Since the aptamer conformation is expected tobe favored in L2bulge6, the presence of theophylline is expected to havelittle or no effect on the conformational dynamics of this switch.L2bulge7 (FIG. 13) was designed to energetically favor the conformation,in which the aptamer structure is not formed and the catalytic core isundisrupted (ribozyme active), by introducing a stabilizing U-A basepair into the stem extending from loop II in this conformation. As thestability of the ribozyme active conformation is significantly higherthan that of the ribozyme inactive conformation for L2bulge7, thepresence of theophylline is expected to have little effect on theconformational dynamics of this ribozyme switch. Ribozyme assaysindicate that L2bulge7 exhibits very low GFP expression levels andL2bulge6 exhibits very high GFP expression levels in the presence andabsence of theophylline (FIG. 14). As rationally designed, bothconstructs exhibit little increase in target expression levels inresponse to theophylline by energetically favoring one of the twoconformational states (FIG. 4B).

L2bulge8 (FIG. 13) was modified from L2bulge7 by replacing the canonicalbase pair (U-A) with a wobble base pair (U-G), thereby reducing thestability of the ribozyme active conformation of L2bulge7 and allowingit to adopt the ribozyme inactive conformation. Similarly, L2bulge9(FIG. 13) was modified in such a way to reduce the energy differencebetween the two conformations of L2bulge7. Ribozyme assays indicate thatL2bulge8 and L2bulge9 exhibit theophylline-dependent up-regulation oftarget gene expression in accordance with the reduced stabilities of theribozyme active conformations and energy differences between the twoadoptable conformations for each of these switch constructs (FIG. 4B andFIG. 14).

In addition, a series of three tuned ‘OFF’ switches were constructed byusing rational energetic tuning strategies from L2bulgeOff 1 as a basestructure. L2bulgeOff2 and L2bulgeOff3 were constructed to demonstratetunability of the ‘OFF’ switch platform using similar energetic designstrategies (FIG. 13). These switch variants exhibit differenttheophylline-responsive dynamic ranges from that of L2bulgeOff1 (FIG. 4Cand FIG. 14).

Flow cytometry analysis of the tuned ribozyme switch series demonstratethat the tuned switches exhibit corresponding shifts in the meanfluorescence of the cell populations in the presence and absence oftheophylline (FIG. 10). The relative dynamic ranges of the switchesacross the full regulatory range bracketed by the ribozyme active andinactive controls, sTRSV and sTRSV Contl respectively, are presented inFIG. 14.

Among the twelve tuned switches (both ‘ON’ and ‘OFF’), the dynamicregulatory ranges of most of these switches are in agreement with ourrational tuning strategies based on the −ΔG and ΔΔG values predicted byRNAstructure 4.2. Two exceptions are noted: L2bulge9 and L2bulgeOff3.L2bulge9 exhibits a larger dynamic regulatory effect despite its higherΔΔG than L2bulge8. L2bulgeOff3 exhibits a smaller dynamic regulatoryeffect despite its smaller ΔΔG than L2bulgeOff2. However, it is moredifficult to make a direct comparison between L2bulgeOff2 andL2bulgeOff3, as both conformations of L2bulgeOff3 are significantly morestable than those of L2bulgeOff2, likely resulting in L2BulgeOff3 lessfrequently switching between its two conformations and thus enablingthis molecule to get ‘trapped’ in its lower free energy states. Inaddition, outliers may also arise because the RNAstructure programpredicts these energy values based on the secondary structure of aparticular conformation and does not take into consideration energycontributions from tertiary interactions (that have been observed inprior work (S2)) in estimating these energies. Nevertheless, wedemonstrate that energetic predictions based solely on secondarystructure are useful for our rational tuning design strategies. Thedifferent dynamic regulatory ranges exhibited by our tuned switches inresponse to their specific effector (FIG. 14) validate that suchresponse programming can be achieved by altering the nucleotidecomposition of the information transmission region within a switch,thereby demonstrating the interdependence between RNA sequence,structure, and function.

Detailed Methods and Materials

-   Plasmid and Switch Construction. The modular plasmid, pRzS, was    constructed and employed as a universal vector for the    characterization of all ribozyme switches. The engineered ribozyme    constructs were generated by PCR amplification using the appropriate    oligonucleotide templates and primers. All oligonucleotides were    synthesized by Integrated DNA Technologies. All engineered ribozyme    constructs were cloned into two unique restriction sites, AvrII and    XhoI, 3 nucleotides downstream of the yEGFP stop codon and upstream    of an ADH1 terminator. Cloned plasmids were transformed into an    electrocompetent Escherichia coli strain, DH10B (Invitrogen) and all    ribozyme constructs were confirmed by subsequent sequencing    (Laragen, Inc). Confirmed plasmid constructs were transformed into    a S. cerevisiae strain (W303 MAT a his3-11,15 trpl-1 leu2-3 ura8 3-1    ade2-1) using standard lithium acetate procedures (S3).-   Ribozyme Characterization Assays. S. cerevisiae cells harboring the    appropriate plasmids were grown in synthetic complete medium    supplemented with an appropriate dropout solution and sugar (2%    raffinose, 1% sucrose) overnight at 30° C. Overnight cultures were    back diluted into fresh medium to an optical density at 600 nm    (OD₆₀₀) of approximately 0.1 and grown at 30° C. An appropriate    volume of concentrated effector stock (to the appropriate final    concentration of theophylline or tetracycline) dissolved in medium    or an equivalent volume of the medium (no effector control) was    added to the cultures at the time of back dilution. In addition, at    this time an appropriate volume of galactose (2% final    concentration) or an equivalent volume of water were added to the    cultures for the induced and non-induced controls, respectively. For    specificity assays, an appropriate volume of a concentrated caffeine    or doxycycline stock (final concentrations of 1 mM and 250 μM,    respectively) was added to a separate culture. Cells were grown to    an OD₆₀₀ of 0.8-1.0 or for a period of approximately 6 h before    measuring GFP levels on a Safire fluorescent plate reader (Tecan)    and/or on a Cell Lab Quanta SC flow cytometer (Beckman Coulter). For    temporal response assays, cultures were grown as described above in    the absence of the appropriate effector and fluorescence data were    taken every 30 min. After 4 h growth, appropriate volumes of the    concentrated effector stock or plain medium were added to the    cultures and fluorescence was monitored for several hours    thereafter.-   Cell Growth Regulation Assays. For liquid culture assays, S.    cerevisiae cells carrying appropriate plasmids were back diluted and    grown according to procedures described above with minor    modifications. A competitive inhibitor of the his5 gene product,    3-amino-triazole (3AT), was added to a final concentration of 5 mM    to increase the threshold level of histidine required for cell    growth. Cultures were grown in various theophylline concentrations    and the growth of each sample was monitored over a 24 h period. The    theophylline-regulated growth at 24 h is reported in terms of OD₆₀₀    readings measured on the Tecan. For plate-based assays, 10 μL of the    back diluted culture samples was streaked on plates containing 0 and    5 mM theophylline. A higher concentration of 3AT (25 mM) was used in    the plate-based assays to optimize visual assessment of    theophylline-regulated cell growth.-   Metabolite Sensing Assays. S. cerevisiae cells carrying appropriate    plasmids were back diluted and grown according to procedures    described above with minor modifications. Cultures were grown in the    absence and presence of xanthosine (250 μM final concentration). To    account for inducer depletion, galactose was added to the cultures    at 8 h time intervals to a 2% final concentration. Fluorescence    levels of the samples were monitored over a 48 h period according to    procedures described above. For HPLC analysis, cell extracts were    prepared after appropriate growth periods following xanthosine    feeding by rapid freezing of cell cultures in liquid nitrogen in the    form of beads. Frozen cell beads were subsequently lysated by    grinding using a mortar and pestle followed by extraction with    methanol. Intracellular metabolite levels were analyzed using an    HPLC system integrated with a mass spectrometer (HPLC-MS) (Agilent    Technologies), which enables confirmation of metabolite peaks based    on their corresponding molecular weights.-   Fluorescence Quantification. The population-averaged fluorescence of    each sample was measured on a Safire fluorescence plate reader with    the following settings: excitation wavelength of 485 nm, an emission    wavelength of 515 nm, and a gain of 100. Fluorescence readings were    normalized to cell number by dividing fluorescence units by the    OD₆₀₀ of the cell sample and subtracting the background fluorescence    level to eliminate autofluorescence. Fluorescence distributions    within the cell populations were measured on a Quanta flow cytometer    with the following settings: 488 nm laser line, 525 nm bandpass    filter, and PMT setting of 5.83. Fluorescence data was collected    under low flow rates for approximately 30,000 cells. Viable cells    were selected and fluorescence levels were determined from 10,000    counts in this selected population. A non-induced cell population    was used to set a ‘negative GFP’ gate. Cells exhibiting fluorescence    above this negative gate are defined as the ‘positive GFP’ cell    population.

Similar to previous reports (S4, S5), we report gene expression levelsas ‘fold’, where 1 fold is defined as the reporter gene expression levelof sTRSV relative to the background fluorescence level. Ligand-directedregulatory effects are reported as fold gene expression levelsnormalized to the levels in the absence of effector. All fluorescencedata and mean±s.d. are reported from at least three independentexperiments.

-   Quantification of Cellular Transcript Levels. cDNA was synthesized    using gene-specific primers and Superscript III Reverse    Transcriptase (Invitrogen) following manufacturer's instructions.    Relative transcript levels were quantified from the cDNA samples by    employing an appropriate primer set and the iQ SYBR Green Supermix    (BioRAD) according to manufacturer's instructions on an iCycler iQ    qRT-PCR machine (BioRAD). The resulting data were analyzed with the    iCycler iQ software according to manufacturer's instructions.    Transcript levels of switch constructs were normalized to that of    the endogenous actI gene (S6) using actI-specific primers. All data    are reported from three independent experiments.

REFERENCES CITED IN EXAMPLE 2

-   S1. Jenison R D, Gill S C, Pardi A, Polisky B (1994) Science    263:1425-9.-   S2. Khvorova A, Lescoute A, Westhof E, Jayasena S D (2003) Nat    Struct Biol 10:708-12.-   S3. Gietz R, Woods R (2002) in Guide to Yeast Genetics and Molecular    and Cell Biology, Part B, eds. Guthrie C, Fink, G (Academic Press,    San Diego, Vol. 350, pp. 87-96).-   S4. Isaacs F J, Dwyer D J, Ding C, Pervouchine D D, Cantor C R,    Collins J J (2004) Nat Biotechnol 22:841-7.-   S5. Yen L, Svendsen J, Lee J S, Gray J T, Magnier M, Baba T, D'Amato    R J, Mulligan R C (2004) Nature 431:471-6.-   S6. Ng R, Abelson J (1980) Proc Natl Acad Sci USA 77:3912-6.

EXAMPLE 3 The Ribozyme Switch Platform Exhibits Portability AcrossDiverse Cellular Systems

The cleavage activity of the hammerhead ribozyme is independent of celltype, as it does not require cell-specific machinery to assist itsself-cleavage, thereby making this regulatory element universallyapplicable to different cellular systems. Therefore, in order todemonstrate that the constructed ribozyme-based switch platform retainsligand-regulated gene control activity in a cellular system differentthan S. cerevisiae, we implemented the strand displacement-based ONswitches (L2bulge1, L2bulge8, and L2bulge9) in mammalian T cells(CTLL-2). Similar to the implementation in yeast, the ribozyme switchconstructs were built by placing these switches downstream (in the 3′UTR, surrounded by spacer/insulater sequences) of a target fusion geneencoding enhanced green fluorescence protein (EGFP) and interleukin-2(IL-2). An increase in target gene expression levels was observed in thepresence of 1 mM theophylline in all ON switches, relative to those inthe absence of effector (FIG. 17), exhibiting a ligand-dependentup-regulation of gene expression by this ON switch in mammalian cellsand demonstrating the switch platform portability across differentcellular systems.

Alternative Strategies for Programming the Dynamic Range of SwitchResponse

The dynamic regulatory response of the ribozyme switch platform can beprogrammed to yield new regulatory response ranges through theexpression of multiple copies of individual, identical switches withinthe 3′ UTR of the target gene. For instance, when these switches wereexpressed in multiple copies, the ligand-regulated dynamic responseranges of these multi-copy switch constructs were observed to bedifferent from those exhibited by single-copy switch constructs.Specifically, the multi-copy constructs exhibit lower expression levelsboth in the absence and presence of effector than those of thecorresponding single-copy constructs due to the independent cleavageactivity of each ribozyme switch, which results in higher over allcleavage activity of the combined system. This tuning method isapplicable in different cellular systems as shown in FIGS. 17 and 18.Therefore, multi-copy expression of a ribozyme switch provides aprogramming method through which to achieve a dynamic regulatoryresponse different from that of the single switch parent, particularlywhen a regulatory system resulting in a low basal expression level isdesired.

EXAMPLE 4 Development of Trans-Ribozymes for In Vivo Utility

We have taken the rules for in vivo activity established forcis-ribozymes and applied them to the development of trans-ribozymes. Wedemonstrate that precise engineering of the intramolecular reactionbetween the ribozyme and target transcript is required for efficienttrans-ribozymes at intracellular Mg²⁺ concentrations. To improve thecorrelation between our in vitro and in vivo assays of ribozymeefficiency, we employed a dual cis-hammerhead ribozyme cassette toexcise our trans-ribozymes designs from the ribonucleoprotein particlein vivo. Additionally, we developed an improved in vitro assay that moreaccurately translates in vitro cleavage efficiency into in vivoregulation of target transcripts. We anticipate that these newlyconstructed fast, efficient trans-hammerhead ribozymes will efficientlyregulate gene expression in vivo.

Ribozymes as Actuators

Ribozymes are RNA molecules that catalyze a variety of chemicalreactions such as self-cleavage or ligation [13]. Various naturallyoccurring ribozymes have been identified in viruses, viroids, andprotozoans. One of the first catalytic RNAs was discovered in thesatellite RNA of the tobacco ring spot viroid (sTRSV) [14]. In vivo thispathogenic viroid was shown to act in cis and self-cleave duringreplication. Since the discovery of the first ribozyme, various classesof natural ribozymes, including hairpin and hammerhead ribozymes, havebeen identified and extensively characterized.

The hammerhead ribozyme (hRz) is one of the most extensively studiedribozymes [13, 15-17]. It is comprised of three helical regions thatconverge on a highly conserved catalytic core of eleven nucleotides(nts) (FIG. 19A) [18, 19]. Cleavage is sequence-specific and targets a5′-NUX-3′ triplet, where N is any base, U is uracil, and X is any baseexcept guanine. The optimal NUX for efficient and fast cleavage is GUC.Ribozyme cleavage is catalyzed when the 2′ hydroxyl group from Xdirectly 3′ of the cleavage site is deprotonated. This nucleophile thenattacks the scissile phosphate and, through a penta-coordinated trigonalbi-pyramidal transition state, produces a 5′ and 3′ product (FIG. 19B)[17].

Folding of the hRz into an active conformation is postulated to proceedthrough dual divalent ion binding events (FIG. 19C). A high affinitybinding event occurs at 500 μM and orders the first set of tertiaryinteractions. The second low affinity addition of ion occurs at 10 mMand restructures the hRz stem orientations such that helix I folds awayfrom helix III and interacts with helix II [16]. HRzs with conservedcatalytic cores that do not maintain specific stem loops are calledminimal hammerhead ribozymes (mhRzs). While mhRzs are active at highdivalent ion concentrations (10 mM), at lower concentrations mhRzs areeffectively inert [14, 18]. Crystal structures of natural hRz depict a“Y”-shaped molecule that has two of the stem loops interacting as“kissing loops” (FIG. 19D) [15]. These tertiary interactions betweenunpaired bases in the stem loops are proposed to stabilize thecatalytically active conformation and obviate high divalent ionconditions. Researchers have demonstrated restored in vitro catalyticactivity at biologically-relevant divalent ion concentrations, between100 and 500 μM, by reincorporating the loops into mhRz designs [14, 18,20-23]. Through elucidation of the design rules for in vivo catalyticactivity, hRz are now poised to be effective regulators of geneexpression.

We have taken the rules established for in vivo activity of cis-hRzs(chRz) and applied them to the development of trans-hRz (thRz). Wedemonstrate additional design constraints for efficient thRz cleavage inengineering the intramolecular binding event between the ribozyme andtarget transcript. In particular, we demonstrate that engineering thestem loops and the length of the targeting arms of the thRz is necessaryto achieve efficient cleavage of the target transcript in vitro. Thiswork represents the first attempt to regulate gene expression via thRzin the model eukaryotic organism S. cerevisiae. In future work, weexpect the newly designed ribozymes to yield the first demonstration ofin vivo regulation of gene expression via thRz. Additionally, thedevelopment of this regulatory platform poises it for the development ofthRz-based ligand-responsive gene regulatory systems, or switches,through the adoption of design rules previously elucidated for chRzs[8].

Initial ThRz Designs Demonstrate In vivo Limitations

ThRz designs were adapted from previous in vitro studies [23] to targeta region in the yEGFP (yeast enhanced green fluorescent protein)transcript (FIGS. 20, 21). While we anticipate that the targeting armswill prove to be amenable to targeting a variety of sequences,developing thRzs that specifically target yEGFP is particularly usefulfor two reasons. First, targeting yEGFP allows us to directly monitorgene expression through fluorescence-based assays. Second, yEGFP is usedto monitor various endogenous proteins via protein fusions. Thus, bydeveloping a ribozyme that targets the expression of yEGFP it will bepossible to regulate the expression of any mRNA tagged with the yEGFPtarget sequence without subsequent redesign of the ribozyme. Theribozyme designs observe the following composition rules. First, thecatalytic core must be conserved to maintain activity (FIG. 20). Second,the target transcript must contain a NUX triplet, where N is any base, Uis uracil, and X is any base but guanine. Finally, for in vivo activity,the catalytically active “Y” shaped conformation must be formed. Toachieve this requirement at physiological Mg²⁺ concentrations, the bulgein stem I and stem loop II, are designed to interact to stabilize theactive conformation. These stem loop sequences are derived from thosefound in the peach latent mosaic viroid (PLMVd) ribozyme, a naturalchRz. Adopting these loops requires careful design considerations whenselecting a target sequence. The bulge region must maintain unpairednucleotides requiring that the corresponding region of the targettranscript does not have sequence complimentary. Additionally, canonicalPLMVd ribozymes exhibit particular stem lengths between the stem I loop,the stem II loop, and the catalytic core. In stem I, 5 base pairsseparate the bulge sequence from the catalytic core. In stem II, 4 basepairs separate the catalytic core and the stem loop sequence. Thus,canonical ribozymes will adopt the following nomenclature “X54” where Xdenotes the number of base pairs formed between the targeting armsequence and the transcript 5′ of the bulge (FIG. 21B).

The initial thRz designs were characterized through in vitro cleavageassays. We modified our cleavage assay from previous examples tofacilitate a more accurate correlation between in vitro results and invivo activity. To develop this method we extended the length of thetarget transcript beyond the region that binds to the targeting arms. Byincluding the peripheral regions, we are able to recapitulate thefolding microenvironment of the full-length transcript for the region ofinterest. By targeting RNA that resembles in vivo transcripts, in vitrocleavage assays are expected to reflect in vivo cleavage efficienciesmore accurately. In these assays both the thRz and 137 nts of the targetgene, yEGFP, were transcribed separately in vitro and subsequentlyincubated together for different time periods to allow cleavage toproceed. Following incubation, cleaved and non-cleaved products werequantified through gel analysis. At 10 mM MgCl₂, the canonicalribozymes, 554 and 654, cleave ˜90% of the target (FIG. 22B). The mRz,SS1, cleaves ˜50% of target. At Mg²⁺ concentrations comparable tophysiological levels, the activity of SS1 is completely abolishedwhereas the canonical ribozymes maintain significant activity.

For this ribozyme to function optimally in vivo as well as in vitro, wemade several alterations. While not wishing to be bound by anyparticular theory, we postulate that ribozymes may encounterinterference in binding to their target transcripts throughcircularization of the ribonucleoprotein particle (RNP) in export fromthe nucleus. The ribozymes are expressed from a pol II promoter. Duringtranscription, a modified nucleotide called the 5′ cap is added to the5′ end of the mRNA transcribed from pol II promoters. As protein factorsassemble onto the cap structure of the transcript, this complex binds toproteins assembled on the poly-A tail and circularizes the transcript.While not wishing to be bound by any particular theory, we postulatethat in the RNP, ribozymes may be inhibited from binding to the targettranscript by the topological constraints imposed by circularization ofthe transcript. Additionally, ribosome loading onto the transcript mayocclude the targeting arms from binding the target transcript.

Implementation of Improved Expression System Facilitates CorrelationBetween In Vitro and in vivo ThRz Cleavage Efficiency

To test this hypothesis, we designed and cloned a flanking chRz cassette(into pCS933 through AvrII and SacII restriction sites) to constructpCS975 (FIG. 23). The cassette contains two chRz, previously shown to behighly efficient at self-cleaving in vivo [14, 18], separated by twounique restriction sites, SacI and SphI, for the rapid cloning of thRzsbetween these two elements.

The chRzs are expected to excise the thRz from the RNP throughself-cleavage resulting in removal of the transcript tails containingthe 5′ cap and the poly-A tail. Removal of the 5′ cap and poly-A tailwill also prevent ribosomes from loading onto this regulatory RNA. Byisolating the liberated transcript from promoter- andterminator-specific transcript tails, the chRz processing cassette willfacilitate thRz portability across a range of expression systems.

In vivo expression of the ribozymes from the redesigned plasmid systemdemonstrated 5-10% knockdown of gene expression (FIG. 24A). To separatethe issues of catalytic efficiency of chRzs and thRzs and the efficiencyof ribozyme targeting in vivo, the ribozymes were transcribed frompCS975 with promoter- and terminator-tails, as well as the chRzcassette. In vitro cleavage assays confirmed that the chRz cleaved withnear perfect efficiency during transcription (data not shown). Theresulting thRzs with chRz tails were incubated with target at variousMg²⁺ concentrations. The thRzs flanked by the chRzs demonstrated loweroverall activity while maintaining similar trends in cleavageefficiencies (FIG. 24B). At high Mg²⁺ concentrations, cleavageefficiency dropped ˜10% compared to the thRzs without the flanking chRzelements. At low Mg²⁺, the thRzs with the chRz cassette cleave ˜5-10% ofthat target which is 30-50% less efficient than the ribozymes withoutthe chRz cassette. These data suggest that the chRz tails that remainedfollowing cis-cleavage interfere with thRz activity. Investigating thefolded sequences in RNAstructure 4.3, a program for determining theexpected secondary structure of RNA molecules, we hypothesized that thechRzs' tails interfered with thRz binding to the target bypreferentially promoting formation of Watson-Crick base paring betweenthe target sequence and the catalytic core. Competition of the coreregion and targeting arms for the target sequence is postulated todecrease the activity of the ribozymes. This effect may be magnified atlow Mg²⁺ concentrations, where proper folding of the ribozyme is notaided by increased levels of this ion. While the in vitro resultsconfirmed the low activity of the thRzs, they also demonstrated that thethRz activity observed in vitro is translated to in vivo regulatoryfunction. Thus, addition of the chRz cassette translates efficienciesdemonstrated in vitro into in vivo results.

The in vitro results from the canonical thRz designs suggest thatefficient cleavage requires high affinity of the targeting arms. At highMg²⁺ concentrations the loop interactions should have little effect onthe rate of cleavage since sufficient Mg²⁺ is present to stabilize thecatalytically active conformation. Thus, observed differences betweenthe mhRz's and the canonical thRzs' cleavage efficiencies at high Mg²⁺may be a result of differing binding affinities. Ribozyme bindingaffinity is expected to correlate with the length of the targeting arms.As length increase, affinity should increase. The targeting arms of SS1are 4 nts and 5 nts shorter than 554 and 654, respectively. At 10 mM,this difference in affinity yields a 40% difference in cleavageefficiency between SS1 and the canonical ribozymes (FIG. 22B). At 500μM, the difference in the length of the targeting arms is magnified.Ribozyme 654, which only differs from 554 by a single nucleotide in thetargeting arms, is ˜25% more efficient. These results strongly suggestthat increasing the length of the targeting arms may significantlyincrease the observed ribozyme cleavage efficiency. Additionally,increasing the targeting arm length should favor proper ribozyme bindingto the target by limiting the competition from the core region bindingto the transcript as shown in RNAstructure 4.3.

Optimization of ThRz Targeting Arms Leads to Fast, Efficient Cleavage atLow MgCl₂ Concentrations

To test this hypothesis, we constructed three new thRz designs withlengthened targeting arms (FIG. 21B). One design increased the length ofthe 5′ arm by 5 nts, 1154. The second (1154+5) and third (1654) designsbuilt on the first, adding 5 nts to the 3′ and 5 nts to the 5′ end,respectively. When folded in RNAstructure 4.3 with the 137 nt targetsequence, the proper ribozyme-binding-to-target fold dominated theenergy landscape as the minimum free energy (MFE) structure and was theonly fold found in the 20 lowest free energy structures. When tested invitro the canonical ribozymes with increased targeting arm length, shownear 100% efficiency at 10 mM Mg²⁺ and greater than 90% at physiologicalMg²⁺ concentrations (FIG. 25). The thRzs with increased targeting armlength exhibit greater cleavage efficiencies at low Mg²⁺ than theprevious canonical thRz design at high Mg²⁺. Furthermore, in vivo assaysindicate that the thRz designs with extended arms (1154, 1154+5, 1654)exhibit greater inhibition of reporter gene expression than the earlierdesigns (FIG. 27). These results indicate that the limiting step in thecleavage event is the intramolecular binding event between the thRz andtarget sequences. Formation of the catalytically active conformationappears to occur readily once the target is found as indicated by thedifference between 654 and 1154. However, increased targeting armlengths may also contribute to increased cleavage efficiency byincreasing the stability of the active tertiary conformation, as well asthe formation of the correct secondary structure.

In vitro cleavage assays demonstrate that lengthening the targeting armsfacilitates rapid cleavage of the target transcript (FIG. 26). Within 10minutes, all of the redesigned thRzs reach greater than 75% cleavageefficiency and one achieves 87%. For in vivo application it is importantthat the ribozymes act on biologically-relevant time scales (˜severalminutes). While previous in vitro studies have demonstrated fast andefficient thRz cleavage at higher Mg²⁺ concentrations (1 mM), theseresults strongly suggest that full-kinetic evaluation will prove thesedesigns to be faster and more efficient even at 500 μM. From initialindications, these may be the fastest thRzs ever reported and by far themost biologically-relevant.

Discussion

Through the rational design of trans-ribozyme targeting arms aided bycomputational models of RNA secondary structures, in vitro thRz activityat intracellular Mg²⁺ concentrations was significantly increased.Additionally, by modifying our in vitro cleavage assay from previousexamples, we have developed a more instructive in vitro assay forevaluating the potential of thRz designs to efficiently cleave targettranscripts in vivo. Also, we have demonstrated that implementation of achRz processing cassette that releases the thRz from the RNP facilitatesimproved correlation between in vitro and in vivo cleavage efficiencies.Finally, the in vitro thRz kinetics at intracellular Mg²⁺ concentrationsdictate that efficient cleavage reactions proceed onbiologically-relevant timescales. With these results in hand, thesubsequent in vivo application of the redesigned thRzs is extremelypromising.

Development of this trans-ribozyme platform represents an opportunity toregulate the expression of target transcripts without the modificationof existing, endogenous cellular components such as binding sites,promoters, repressors, and other cis-acting regulatory elements. Bydeveloping control elements that modulate gene expression withoutmodification of the natural context of the target system, more powerfuland less-invasive cellular programming strategies can be developed.Obviating the need to modify cis-regulatory elements will advancecontrol strategies that can be more quickly transferred tomulti-cellular organisms where such targeted genetic manipulation isoften difficult.

The systems and methods described herein enable the development andcharacterization of thRz switches as control elements that regulate geneexpression of target proteins in response to exogenous and endogenousligands, using the rational coupling of aptamers and thRz domains. Suchligand-controlled riboregulators are termed ribozyme switches. TheSmolke laboratory has recently developed and demonstrated a modular andextensible framework for engineering in vivo ligand-regulated ribozymeswitches [8]. The described switch platforms contain three distinctfunctional domains: a ligand-binding or sensor domain, comprised of anaptamer sequence, a regulatory or actuator domain, comprised of a chRzsequence, and an information transmission domain, comprised of asequence that transmits information between the sensor and the actuatordomain (FIG. 2A). Small molecule-dependent regulation of gene expressionwas demonstrated on various heterologous genes. The platforms enabledthe construction of riboswitches exhibiting up- and down-regulation oftarget expression levels (FIGS. 2B, C). The constructed cis-riboswitchesrepresent a versatile platform for implementing ligand-controlled generegulation in various applications including the programming of cellularcontrol strategies.

ThRz switches offer a significant advantage over cis control strategiesbecause they are able to target endogenous proteins without modifyingthe natural context of the target gene. This is advantageous becausealtering a cell's genome can often be difficult and result in off-targeteffects. The emergence of ligand-responsive control elements that act intrans significantly improve our ability to study natural metabolic andregulatory networks, as well as impose exogenous control and engineernew connections into these systems. By providing a modular interfacebetween engineered gene networks and endogenous circuitry, thesetechnologies significantly advance our ability to program cellularbehavior.

Materials and Methods

-   Plasmid and Ribozyme Construction. The thRz expression construct,    pCS933, was constructed using standard molecular biology techniques    [31]. Briefly, two sets of TEF promoters and CYC1 terminators with    intervening unique restriction sites were cloned into the plasmid    backbone. A version of RFP, tdimer2, was cloned into the SalI and    NotI restriction sites downstream of the second TEF promoter and    served as a transformation control signal. The pCS933 engineered    ribozyme constructs were generated by cloning the appropriate thRz    constructs into the unique restriction sites, AvrII and SacII,    downstream of the first TEF promoter. The thRz with flanking chRzs    construct, pCS975, was constructed by cloning a cassette containing    two chRzs with two intervening unique restriction sites, SphI and    SacI, into AvrII and SalI. The pCS975 engineered ribozyme constructs    were generated by cloning the appropriate thRz constructs into the    SphI and SacI sites. All oligonucleotides were synthesized by    Integrated DNA Technologies (Coralville, Iowa). Cloned plasmids were    transformed into an electrocompetent E. coli strain, DH10B    (Invitrogen, Carlsbad, Calif.), and all cloned ribozyme constructs    were confirmed by sequencing (Laragen, Los Angeles, Calif.).    Confirmed plasmid constructs were transformed into a S. cerevisiae    strain W303 harboring a chromosomally-integrated GFP target    construct (MATa his3-11,15 trpl-1 leu2-3 ura3-1 ade2-1,    pTEF-yEGFP-PEST) using standard lithium acetate procedures [32].-   In vitro cleavage assay. All ribozymes were PCR amplified from their    plasmids along with the 23 nts upstream transcribed from the TEF    promoter. A 137 nt region of the target yEGFP sequence was amplified    by PCR. The forward primer in each of these amplification reactions    harbors the T7 polymerase sequence at its 5′ end such that a fusion    between the desired sequence and the T7 promoter region is    constructed. Sequences were transcribed using an Ampliscribe T7 kit    (Epicentre Technologies, Madison, Wis.) and transcription reactions    were purified through a NucAway column (Ambion, Foster City,    Calif.), following manufacturer's instructions. The target yEGFP    sequence was labeled with [α-³²P]-GTP. Cleavage reactions were    performed at 50 mM Tris-Cl pH 7.0, 100 mM NaCl, and the specified    MgCl₂ concentration. The ratio of ribozyme to target was 10:1 and    7:1 as specified. Reactions were incubated at 95° C. for 5 min,    cooled at room temp for 15 min, and placed for 1 hr at 37° C.    Reactions were quenched with the addition of RNA loading buffer,    heated at 65° C. for 5 min, and chilled at 4° C. for 5 min. Reaction    products were separated on a denaturing PAGE gel and visualized on a    phosphorimager (BioRAD).-   Ribozyme Characterization. S. cerevisiae cells harboring the    appropriate plasmids were grown in synthetic complete medium    supplemented with an appropriate dropout solution and sugar [2%    (wt/vol) dextrose] overnight at 30° C. Overnight cultures were    back-diluted into fresh medium to an optical density at 600 nm    (OD₆₀₀) of ˜0.1 and grown at 30° C. Cells were grown to an OD₆₀₀ of    0.8-1.0 or for a period of ˜6 hr before measuring GFP levels on a    Cell Lab Quanta SC flow cytometer (Beckman Coulter, Fullerton,    Calif.).-   Fluorescence Quantification. Populations average fluorescence values    were measured on a Quanta flow cytometer with the following    settings: 488-nm laser line, 525-nm bandpass filter, and    photomultiplier tube setting of 7.53 on FL1 (GFP) and 6.53 on FL3    (RFP). Fluorescence data were collected under low flow rates for    ˜20,000 viable cells. Cells bearing plasmids not expressing RFP were    used to set a “RFP negative” gate. Viable cells bearing the plasmid    were selected by gating for cells with fluorescence values on FL3    greater than the RFP negative gate. Fluorescence levels were    determined from 10,000 counts in this selected population. Since the    pTEF-yEGFP cassette is integrated into the chromosome, the FL1    values from entire population of viable, RFP positive cells were    averaged to calculate the sample's GFP fluorescence value. All    fluorescence data and mean±SD are reported from at least three    independent experiments.

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Uhlenbeck, Self-cleaving catalytic RNA.    Faseb J, 1993. 7(1): p. 25-30.-   14. De la Pena, M., S. Gago, and R. Flores, Peripheral regions of    natural hammerhead ribozymes greatly increase their self-cleavage    activity. Embo J, 2003. 22(20): p. 5561-70.-   15. Pley, H. W., K. M. Flaherty, and D. B. McKay, Three-dimensional    structure of a hammerhead ribozyme. Nature, 1994. 372(6501): p.    68-74.-   16. Hammann, C., D. G. Norman, and D. M Lilley, Dissection of the    ion-induced folding of the hammerhead ribozyme using ¹⁹F NMR. Proc    Natl Acad Sci USA, 2001.-   98(10): p. 5503-8.-   17. Blount, K. F. and O. C. Uhlenbeck, The structure-function    dilemma of the hammerhead ribozyme. Annu Rev Biophys Biomol    Struct, 2005. 34: p. 415-40.-   18. Khvorova, A., et al., Sequence elements outside the hammerhead    ribozyme catalytic core enable intracellular activity. Nat Struct    Biol, 2003. 10(9): p. 708-12.-   19. Salehi-Ashtiani, K. and J. W. Szostak, In vitro evolution    suggests multiple origins for the hammerhead ribozyme. Nature, 2001.    414(6859): p. 82-4.-   20. Canny, M. D., et al., Fast cleavage kinetics of a natural    hammerhead ribozyme. J Am Chem Soc, 2004. 126(35): p. 10848-9.-   21. Penedo, J. C., et al., Folding of the natural hammerhead    ribozyme is enhanced by interaction of auxiliary elements.    Rna, 2004. 10(5): p. 880-8.-   22. Saksmerprome, V., et al., Artificial tertiary motifs stabilize    trans-cleaving hammerhead ribozymes under conditions of    submillimolar divalent ions and high temperatures. Rna, 2004.    10(12): p. 1916-24.-   23. Weinberg, M. S, and J. J. Rossi, Comparative single-turnover    kinetic analyses of trans-cleaving hammerhead ribozymes with    naturally derived non-conserved sequence motifs. FEBS Lett, 2005.    579(7): p. 1619-24.-   24. Burke, D. H. and S. T. Greathouse, Low-magnesium, trans-cleavage    activity by type III, tertiary stabilized hammerhead ribozymes with    stem 1 discontinuities. BMC Biochem, 2005. 6: p. 14.-   25. Elion, E. A., The Step 5p scaffold. J Cell Sci, 200 1. 114(Pt    22): p. 3967-78.-   26. Park, S. H., A. Zarrinpar, and W. A. Lim, Rewiring MAP kinase    pathways using alternative scaffold assembly mechanisms.    Science, 2003. 299(5609): p. 1061-4.-   27. Liu, L., et al., Sorafenib blocks the RAF/MEK/ERK pathway,    inhibits tumor angiogenesis, and induces tumor cell apoptosis in    hepatocellular carcinoma model PLC/PRF/5. Cancer Res, 2006.    66(24): p. 11851-8.-   28. Shapiro, P., Discovering new MAP kinase inhibitors. Chem    Biol, 2006. 13(8): p. 807-9.-   29. Dominguez, C., D. A. Powers, and N. Tamayo, p38 MAP kinase    inhibitors: many are made, but few are chosen. Curr Opin Drug Discov    Devel, 2005. 8(4): p. 421-30.-   30. Dambach, D. 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EXAMPLE 5 Design Parameters for Allosteric Trans-Hammerhead Ribozyme(ThRz) Switch Platforms

Catalytic Core

The catalytic core of the hammerhead ribozyme consists of twelveconserved nucleotides. From stem I, the sequence follows from stem 1 to5′-CTAGATGAG (SEQ ID NO: 31)-stem II sequence-CGAA-3′ ending at stemIII. The guanine located 5′ of the intervening stem II sequence forms aWatson-Crick base pair (bp) with the cytosine that is 3′ of theintervening stem II sequence. This pairing isolates the catalytic corefrom the stem II sequence.

Catalytic Core: 5′-CTAGATGAG(SEQ ID NO: 31)-stem II sequence-CGAA-3′Target Sequence and Targeting Arms

The target sequence can be of any length, however, the region ofinterest for design purposes is at least 26 by long. (Note: Where targetand targeting arm lengths are defined these values represent a minimum.The length of the sequence will varying based on the GC content of thesequence surround the NUX triplet. Additionally, it is important toengineer the targeting arms to favor proper target-to-ribozyme binding.)Specifically, there are at least 15 bases 3′ of the NUX triplet and atleast 7 bases that are 5′ of it. These bases of interest shall bespecified T1 through T26, where T denotes the base specified in thetarget. Target sequences must contain a NUX triplet at T8-T10, where Nrepresents any base, U represents uracil, and X stands for any base butguanine. For greatest efficiency, choose X to be cytosine and N to beguanine, GUC. Thus the sequence is specified so far as 5′-N₇-GUC—N₁₅-3′.Since the targeting arms identify targets through Watson-Crick basepairing, the NUX triplet specifies bases in the targeting arm. The 3′targeting arm of the ribozyme is immediately 3′ of the ribozymecatalytic core and specified by at least 9 bases. These bases must becomplementary to the T1-T9 such that the 3′ targeting arm is specifiedas: 5′-AC-N₇-3′. The 5′ arm is designed to by with T11-T26, but hasadditional bases denoted as bulge bases, B. Such that the 5′ targetingarm is as follows: 5′-N₁₁—BBB—N₅-3′. Typically, BBB will be UAA a bulgesequence derived from the naturally occurring ribozyme in the PeachLatent Mosaic Viroid. (Note: if stem loops are derived from alternativenatural ribozymes it will be necessary to alter the design rules for thetargeting arm and target sequence.) The bulge is critical forinteraction of stem I and stem II, which stabilizes the catalyticallyactive conformation under physiological magnesium concentrations. Thiswill be discussed more thoroughly as the design of stem II is described.The targeting arms comprise the ribozyme's contribution to the stem Iand stem III region. Additionally, since it is important for the targettranscript not to pair with the bulge bases, the bases T16-T18 must bespecified as K′K′L′ where K′ is any base but uracil and L′ is any basebut adenine. Note that the cytosine at T10 is not base-paired and thisis essential for catalytic activity.

Target Sequence: 5′-N₇-GUC-N₅-K′K′L′-N₈-3′ Target Arms:5′-N₁₁-UAA-N₅-Catalytic core and stem II-AC N₇-3′Stem I

Stem I is comprised of the target transcript based paired to ribozymetargeting arm. This base pairing leads to a helical three-dimensionalstructure and often stem I is called helix I. There are at least 16 byin this helix and 3 free bases.

Stem III

Stem III is comprised of the target transcript based paired to ribozymetargeting arm. This base pairing leads to a helical three-dimensionalstructure and often stem III is called helix III. This helix is at least9 by long.

Stem II

For allosteric control, the proper design of stem II is essential.Additionally, the correct specification of the bulges, helical regions,and aptamer domains is critical for in vivo activity and efficientribozyme cleavage.

Helical Region and Bulge

The helical region proceeds directly 5′ and 3′ from the catalytic core.The catalytic core ends with base pairing between the 5′G and the 3′C.From this base paired closure of the core, the stem continues with 4additional bp. This value is flexible depending on the bulge sequenceschosen. 4 is optimal for the bulges derived from the Peach Latent MosaicViroid (PLMVd) ribozyme. The bulge in stem II is required for in vivoactivity. The interaction between the bulge bases in stem I and stem IIbrings the stems into closer proximity of each other and stabilizes thecatalytically active ribozyme conformation at low magnesiumconcentration, such as persists under in vivo conditions. The bulgeconsists of 6 bases, 3 on each side of the helix. From the 5′ helicalregion the sequence for the PLMVd bulge is: UAG and from the 3′ helicalregion: UAA.

Stem II: 5′ Catalytic Core-N₄-UAG-Aptamer Region-UAA-N₄-CatalyticCore-3′

Aptamer Region

Following the bulge, another helical region follows. This helical regionis designed to switch between two conformations which are dependent uponligand binding at the aptamer domain. The design of this region iscritical for the development of ligand-regulated switches whichstandardize and amplify the conformational change associated with theligand binding event by pairing aptamer and expression platform domainsthrough a linker platform (information transmission domain). In theseengineered switches, conformational changes associated with ligandbinding to the aptamer domain are transduced to a common larger-scaleconformational rearrangement in the ribozyme catalytic core, therebyregulating its conversion between an active and inactive state. Twotypes of information transmission domains are used for connecting theaptamer domain to stem II; communication modules act through helixslipping mechanism and strand displacement linkers act through stranddisplacement.

Communication Module

When the communication module is the linker chosen to connect theaptamer to stem II, the precise sequence that yields switching behaviormust be chosen uniquely for each aptamer through iterative designchoices. Ideally, binding at the aptamer changes base pairing throughthe communication module, which in turn induces a base-pair “slip”through the stem II region into the catalytic core. This slippingmechanism regulates the conversion between the active and inactiveconformations.

Strand Displacement

When strand displacement is the method of choice for connecting theaptamer to stem II, this displacement can be rationally designed toaccompany an array of aptamers. Strand displacement induces a largerscale change in the stem II region upon ligand binding. This largerscale conformation change makes the mechanism of transducing ligandbinding into conformation changes in the ribozyme more robust.

Aptamers

A wide array of aptamers can be coupled to the stem II region viavarious linkers. Input ligands include small molecules, proteins, andoligonucleotides. Theoretically, an aptamer can be developed to nearlymolecule through SELEX. This makes the trans-ribozyme switch platformsextremely flexible in their range of input ligands.

Stem II: 5′-Helix II-Bulge5′-linker5′-Aptamer sequence-linker3′-Bulge3′-Helix 11-3′

Steric Hindrance Considerations

Since trans-ribozyme switch platforms act through a bimolecular reactionit is important to ensure that the tails of the transcribed ribozyme donot occlude the targeting arms. Additionally, transcription tails shouldnot possess significant secondary structures that impede formation ofthe catalytically active conformation of the ribozyme. To eliminate,hairpins and other secondary structures from interfering with targetbinding or core formation, cis-acting ribozymes can be placed 3′ and 5′of the trans-riboswitch platform to trim the transcript. This will yieldthe trans-ribozyme switch platform with smaller, precise tails.Additionally, the cis-ribozyme cassette isolates the trans-ribozyme fromthe specific sequences of the promoter and terminator. Thus, the modulebecomes more predictably portable across a range of expression systems.Note that it is necessary to design sequence elements outside thetrans-riboswitch platform to ensure the platform is able to efficientlycleave target.

Elements Outside of Platform

With Transcript Tails:

5′-Transcript tail-trans-ribozyme switch platform-Transcript tail-3′

With Flanking Cis-Ribozymes:

Before Cleavage of Cis-Ribozymes. 5′-Transcript tail-5′cis-ribozyme-trans-ribozyme switch platform-3′ cis-ribozyme-Transcripttail-3′

After Cleavage of Cis-Ribozymes. 5′-Several bases-trans-ribozyme switchplatform-a few bases-3′ (The dashes above generally representrestriction enzyme sites.)

Final Trans-Ribozyme Switch Platform

5′-N₁₁-UAA-N₅-CTAGATGAG (SEQ ID NO: 31)-N₄-UAG-linker5′-Aptamer-linker 3′-UAA-N₄-CGAA-AC N₇-3′Final Target Sequence Requirements

5′-N₇-GUC-N₅-K′K′L′-N₈-3′

The invention claimed is:
 1. An aptamer-regulated ribozyme for cleavinga target nucleic acid, comprising (a) a trans-acting hammerhead ribozymecomprising catalytic core, a 5′ targeting arm which hybridizes to a 3′sequence of said target nucleic acid, a 3′ targeting arm whichhybridizes to a 5′ sequence of said target nucleic acid, and a stemduplex region extending from said catalytic core with a single-strandedloop region opposite to said catalytic core; (b) an informationtransmission domain (ITD) having a first end and a second end, whichinformation transmission domain is directly coupled to said loop throughsaid first end; and (c) an aptamer coupled to said informationtransmission domain through said second end, said aptamer binds to aligand, wherein said ITD is between the trans-acting hammerhead ribozymeand the aptamer, and comprises: (i) a general transmission region, (ii)a switching strand that hybridizes with the general transmission regionin the absence of the ligand, and, (iii) a competing strand thathybridizes with the general transmission region in the presence of theligand, wherein the switching strand and the competing strand compete tobind to the general transmission region through hybridizationinteractions, wherein binding of said ligand to said aptamer favors aconformation change in the aptamer, wherein said conformation changecauses the ITD to favor the binding of the general transmission regionto said competing strand via a strand-displacement mechanism; and,wherein hybridization between the general transmission region and thecompeting strand causes, via the interaction of said informationtransmission domain with one or more of said loop, said stem or saidcatalytic core, said aptamer-regulated ribozyme to cleave the targetnucleic acid at a rate dependent upon the presence or absence of saidligand.
 2. The aptamer-regulated ribozyme of claim 1, wherein binding ofsaid ligand to said aptamer alters the size of said loop, therebyaltering the ability of said aptamer-regulated ribozyme to cleave saidtarget nucleic acid in a manner dependent on said ligand.
 3. Theaptamer-regulated ribozyme of claim 1, wherein binding of said ligand tosaid aptamer alters the size of said catalytic core, thereby alteringthe ability of said aptamer-regulated ribozyme to cleave said targetnucleic acid in a manner dependent on said ligand.
 4. Theaptamer-regulated ribozyme of claim 1, wherein the ligand is a smallmolecule having a molecular weight less than 2500 amu and/or is cellpermeable.
 5. The aptamer-regulated ribozyme of claim 1, wherein theligand is a metal ion.
 6. The aptamer-regulated ribozyme of claim 1,wherein the ligand is a natural product.
 7. The aptamer-regulatedribozyme of claim 6, wherein the ligand is a signal transduction secondmessenger molecule.
 8. The aptamer-regulated ribozyme of claim 6,wherein the ligand is a post-translationally modified protein.
 9. Theaptamer-regulated ribozyme of claim 1, wherein the ligand is selectedfrom the group consisting of polypeptides, peptides, nucleic acids,carbohydrates, fatty acids and lipids, a non-peptide hormone andmetabolic precursors or products thereof.
 10. The aptamer-regulatedribozyme of claim 1, wherein the ligand is selected from the groupconsisting of enzyme co-factors, enzyme substrates and products ofenzyme-mediated reactions.
 11. An expression construct comprising (i) acoding sequence which, when transcribed to RNA, produces saidaptamer-regulated ribozyme of claim 1, and (ii) one or moretranscriptional regulatory sequences that regulate transcription of saidRNA in a cell containing said expression construct.
 12. A method forrendering expression of a target gene in a cell dependent on thepresence or absence of a ligand, comprising introducing into the cell atrans-acting hammerhead ribozyme comprising: (a) a catalytic core, a 5′targeting arm which hybridizes to a 3′ sequence of an mRNA transcribedfrom said target gene, a 3′ targeting arm which hybridizes to a 5′sequence of said mRNA transcribed from said target gene, and a stemduplex region extending from said catalytic core with a single-strandedloop region opposite to said catalytic core; (b) an informationtransmission domain (ITD) having a first end and a second end, whichinformation transmission domain is directly coupled to said loop throughsaid first end; and (c) an aptamer coupled to said informationtransmission domain through said second end, said aptamer binds to aligand; wherein said ITD is between the trans-acting hammerhead ribozymeand the aptamer, and comprises: (i) a general transmission region, (ii)a switching strand that hybridizes with the general transmission regionin the absence of the ligand, and, (iii) a competing strand thathybridizes with the general transmission region in the presence of theligand, wherein the switching strand and the competing strand compete tobind to the general transmission region through hybridizationinteractions, wherein binding of said ligand to said aptamer favors aconformation change in the aptamer, wherein said conformation changecauses the ITD to favor the binding of the general transmission regionto said competing strand via a strand-displacement mechanism; and,wherein hybridization between the general transmission region and thecompeting strand causes, via the interaction of said informationtransmission domain with one or more of said loop, said stem or saidcatalytic core, said trans-acting hammerhead ribozyme to cleave the mRNAtranscribed from said target gene at a rate dependent upon the presenceor absence of said ligand so as to render expression of the target genein the cell dependent on said ligand.
 13. The method of claim 12,wherein the ligand is produced by said cell.
 14. The method of claim 12,wherein the ligand is a cell permeable agent that is contacted with saidcell.
 15. A method of determining the amount of an analyte in a cellwhich expresses a reporter gene, comprising: (i) introducing into thecell a trans-acting hammerhead ribozyme comprising (a) a catalytic core,a 5′ targeting arm which hybridizes to a 3′ sequence of an mRNAtranscribed from said reporter gene, a 3′ targeting arm which hybridizesto a 5′ sequence of said mRNA transcribed from said reporter gene, and astem duplex region extending from said catalytic core with asingle-stranded loop region opposite to said catalytic core; (b) aninformation transmission domain (ITD) having a first end and a secondend, which information transmission domain is directly coupled to saidloop through said first end; and (c) an aptamer coupled to saidinformation transmission domain through said second end, said aptamerselectively binds to said analyte; wherein said ITD is between thetrans-acting hammerhead ribozyme and the aptamer, and comprises: (i) ageneral transmission region, (ii) a switching strand that hybridizeswith the general transmission region in the absence of the ligand, and,(iii) a competing strand that hybridizes with the general transmissionregion in the presence of the ligand, wherein the switching strand andthe competing strand compete to bind to the general transmission regionthrough hybridization interactions, wherein binding of said analyte tosaid aptamer favors a conformation change in the aptamer, wherein saidconformation change causes the ITD to favor the binding of the generaltransmission region to said competing strand via a strand-displacementmechanism; and, wherein hybridization between the general transmissionregion and the competing strand causes, via the interaction of saidinformation transmission domain with one or more of said loop, said stemor said catalytic core, said trans-acting hammerhead ribozyme to cleavethe mRNA transcribed from said reporter gene at a rate dependent uponthe presence or absence of said analyte; (ii) measuring the amount ofexpression of said reporter gene; and (iii) correlating the amount ofexpression of said reporter gene with the amount of analyte, therebydetermining the amount of the analyte in the cell.
 16. A method forcausing phenotypic regulation of cell growth, differentiation orviability in cells of a patient, comprising introducing into cells insaid a patient an aptamer-regulated ribozyme of claim 1, where saidaptamer binds to a ligand, the concentration of which is dependent oncellular phenotype, wherein binding of said ligand to said aptamerinduces a conformational change between active and inactive forms ofsaid trans-acting hammerhead ribozyme, and said active form of saidtrans-acting hammerhead ribozyme inhibits expression of said target geneto alter the regulation of cell growth, differentiation or viability insaid cells.
 17. A pharmaceutical preparation comprising anaptamer-regulated ribozyme of claim 1, or an expression construct which,when transcribed, produces an RNA including said aptamer-regulatedribozyme, and a pharmaceutically acceptable carrier suitable for useadministration to a human or nonhuman patient.